Vitamin C Inhibits Lipopolysaccharide-Induced Hyperinflammatory State of Chronic Myeloid Leukemia Cells through Purinergic Signaling and Autophagy.

Background: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the overproduction of white blood cells, leading to symptoms such as fatigue, infections, and other complications. CML patients must take measures to prevent infections to mitigate the exacerbation of cancer cell proliferation and comorbidities. Methods: This study investigated whether vitamin C can suppress the hyperinflammatory activation of K-562 cells induced by lipopolysaccharide (LPS) and whether purinergic signaling (ATP and P2X7 receptor) and autophagy play a role in it. Two different doses of vitamin C (5 µg/mL and 10 µg/mL) were employed, along with the lysosome inhibitor chloroquine (CQ; 100 µM), administered 2 h prior to LPS stimulation (10 ng/mL) for a duration of 22 h in K-562 cells (3 × 105 cells/mL/well). Results: Both doses of vitamin C reduced the release of interleukin-6 (IL-6) (5 µg/mL, p < 0.01 and 10 µg/mL, p < 0.01) and tumor necrosis factor (TNF) (5 µg/mL, p < 0.01 and 10 µg/mL, p < 0.01) induced by LPS. Furthermore, in LPS + CQ-stimulated cells, vitamin C at a concentration of 10 µg/mL inhibited the expression of LC3-II (p < 0.05). Conversely, both doses of vitamin C led to the release of the anti-inflammatory cytokine interleukin-10 (IL-10) (5 µg/mL, p < 0.01 and 10 µg/mL, p < 0.01), while only the 10 µg/mL dose of vitamin C induced the release of Klotho (10 µg/mL, p < 0.01). In addition, both doses of vitamin C reduced the accumulation of ATP (5 µg/mL, p < 0.01 and 10 µg/mL, p < 0.01) and decreased the expression of the P2X7 receptor at the mRNA level. Conclusions: Vitamin C inhibits the hyperinflammatory state induced by LPS in K-562 cells, primarily by inhibiting the ATP accumulation, P2X7 receptor expression, and autophagy signaling.

Palladium (II) compounds containing oximes as promising antitumor agents for the treatment of osteosarcoma: An in vitro and in vivo comparative study with cisplatin.

Drug resistance, evasion of cell death and metastasis are factors that contribute to the low cure rate and disease-free survival in osteosarcomas (OS). In this study, we demonstrated that a new class of oxime-containing organometallic complexes called Pd-BPO (O3) and Pd-BMO (O4) are more cytotoxic than cisplatin (CDDP) for SaOS-2 and U2OS cells using the MTT assay. Annexin-FITC/7-AAD staining demonstrated a greater potential for palladium-oxime complexes to induce death in SaOS-2 cells than CDDP, an event confirmed using the pan-caspase inhibitor Z-VAD-FMK. Compared to CDDP, only palladium-oxime complexes eradicated the clonogenicity of SaOS-2 cells after 7 days of treatment. The involvement of the lysosome-mitochondria axis in the cell death-inducing properties of the complexes was also evaluated. Using LysoTracker Red to label the acidic organelles of SaOS-2 cells treated with the O3 and O4 complexes, a decrease in the fluorescence intensity of this probe was observed in relation to CDDP and the control. Lysosomal membrane permeabilization (LMP) was also induced by the O3 and O4 complexes in an assay using acridine orange (A/O). The greater efficiency of the complexes in depolarizing the mitochondrial membrane compared to SaOS-2 cells treated with CDDP was also observed using TMRE (tetramethyl rhodamine, ethyl ester). For in vivo studies, C. elegans was used and demonstrated that both complexes reduce body bends and pharyngeal pumping after 24 h of treatment to the same extent as CDDP. We conclude that both palladium-oxime complexes are more effective than CDDP in inducing tumor cell death. The toxicity of these complexes to C. elegans was like that induced by CDDP. These results encourage preclinical studies aimed at developing more effective drugs for the treatment of osteosarcoma (OS). Furthermore, we propose palladium-oxime complexes as a new class of antineoplastic agents.

Artemisia vulgaris Induces Tumor-Selective Ferroptosis and Necroptosis via Lysosomal Ca2+ Signaling.

OBJECTIVE: To evaluate the chemical composition and effects of Artemisia vulgaris (AV) hydroalcoholic extract (HEAV) on breast cancer cells (MCF-7 and SKBR-3), chronic myeloid leukemia (K562) and NIH/3T3 fibroblasts. METHODS: Phytochemical analysis of HEAV was done by high-performance liquid chromatography-mass (HPLC) spectrometry. Viability and cell death studies were performed using trypan blue and Annexin/FITC-7AAD, respectively. Ferrostatin-1 (Fer-1) and necrostatin-1 (Nec-1) were used to assess the mode of HEAV-induced cell death and acetoxymethylester (BAPTA-AM) was used to verify the involvement of cytosolic calcium in this event. Cytosolic calcium measurements were made using Fura-2-AM. RESULTS: HEAV decreased the viability of MCF-7, SKBR-3 and K562 cells (P<0.05). The viability of HEAV-treated K562 cells was reduced compared to HEAV-exposed fibroblasts (P<0.05). Treatment of K562 cells with HEAV induced cell death primarily by late apoptosis and necrosis in assays using annexin V-FITC/7-AAD (P<0.05). The use of Nec-1 and Fer-1 increased the viability of K562 cells treated with HEAV relative to cells exposed to HEAV alone (P<0.01). HEAV-induced Ca2+ release mainly from lysosomes in K562 cells (P<0.01). Furthermore, BAPTA-AM, an intracellular Ca2+ chelator, decreased the number of non-viable cells treated with HEAV (P<0.05). CONCLUSIONS: HEAV is cytotoxic and activates several modalities of cell death, which are partially dependent on lysosomal release of Ca2+. These effects may be related to artemisinin and caffeoylquinic acids, the main compounds identified in HEAV.

Blocking Autophagy by the Two-Pore Channels Antagonist Tetrandrine Improves Sorafenib-Induced Death of Hepatocellular Carcinoma Cells.

Sorafenib, an oral multi-kinase inhibitor, used to treat hepatocellular carcinoma (HCC). However, drug resistance is still common in several HCC patients. This complex mechanism is not yet fully elucidated, driving the search for new therapeutic targets to potentiate the antitumoral effect of sorafenib. Recent findings have linked the expression of Two-Pore Channels (TPCs) receptors with the development and progression of cancer. TPCs receptors are stimulated by NAADP, a Ca2+ messenger, and inhibited by their antagonists Ned-19 and tetrandrine. Here, we investigate the participation of TPCs inhibition in cell death and autophagy in sorafenib-treated HCC cells. Here, we show that the association of sorafenib with tetrandrine increased sorafenib-induced cell death accompanied by increased lysotracker fluorescence intensity. In contrast, these effects were not observed after treating these cells with Ned-19. The pharmacological TPC antagonists by Ned-19 and tetrandrine or siRNA-mediated TPC1/2 inhibition decreased sorafenib-induced Ca2+ release, reinforcing the participation of TPCs in sorafenib HCC responses. Furthermore, the association tetrandrine and sorafenib blocked autophagy through ERK1/2 pathway inhibition, which represents a putative target for potentiating HCC cell death. Therefore, our study proposes the use of tetrandrine analogs with the aim of improving sorafenib therapy. Also, our data also allow us to suggest that TPCs may be a new target in anticancer therapies.

NAADP-Evoked Ca2+ Signaling Leads to Mutant Huntingtin Aggregation and Autophagy Impairment in Murine Astrocytes.

Huntington's disease (HD) is a progressive neurodegenerative disease characterized by mutations in the huntingtin gene (mHtt), causing an unstable repeat of the CAG trinucleotide, leading to abnormal long repeats of polyglutamine (poly-Q) in the N-terminal region of the huntingtin, which form abnormal conformations and aggregates. Alterations in Ca2+ signaling are involved in HD models and the accumulation of mutated huntingtin interferes with Ca2+ homeostasis. Lysosomes are intracellular Ca2+ storages that participate in endocytic and lysosomal degradation processes, including autophagy. Nicotinic acid adenine dinucleotide phosphate (NAADP) is an intracellular second messenger that promotes Ca2+ release from the endo-lysosomal system via Two-Pore Channels (TPCs) activation. Herein, we show the impact of lysosomal Ca2+ signals on mHtt aggregation and autophagy blockade in murine astrocytes overexpressing mHtt-Q74. We observed that mHtt-Q74 overexpression causes an increase in NAADP-evoked Ca2+ signals and mHtt aggregation, which was inhibited in the presence of Ned-19, a TPC antagonist, or BAPTA-AM, a Ca2+ chelator. Additionally, TPC2 silencing revert the mHtt aggregation. Furthermore, mHtt has been shown co-localized with TPC2 which may contribute to its effects on lysosomal homeostasis. Moreover, NAADP-mediated autophagy was also blocked since its function is dependent on lysosomal functionality. Taken together, our data show that increased levels of cytosolic Ca2+ mediated by NAADP causes mHtt aggregation. Additionally, mHtt co-localizes with the lysosomes, where it possibly affects organelle functions and impairs autophagy.

Cannabidiol Recovers Dopaminergic Neuronal Damage Induced by Reserpine or α-synuclein in Caenorhabditis elegans.

Progressive neurodegenerative disorders such as Parkinson Disease (PD) lack curative or long-term treatments. At the same time, the increase of the worldwide elderly population and, consequently, the extension in the prevalence of age-related diseases have promoted research interest in neurodegenerative disorders. Caenorhabditis elegans is a free-living nematode widely used as an animal model in studies of human diseases. Here we evaluated cannabidiol (CBD) as a possible neuroprotective compound in PD using the C. elegans models exposed to reserpine. Our results demonstrated that CBD reversed the reserpine-induced locomotor alterations and this response was independent of the NPR-19 receptors, an orthologous receptor for central cannabinoid receptor type 1. Morphological alterations of cephalic sensilla (CEP) dopaminergic neurons indicated that CBD also protects neurons from reserpine-induced degeneration. That is, CBD attenuates the reserpine-induced increase of worms with shrunken soma and dendrites loss, increasing the number of worms with intact CEP neurons. Finally, we found that CBD also reduced ROS formation and α-syn protein accumulation in mutant worms. Our findings collectively provide new evidence that CBD acts as neuroprotector in dopaminergic neurons, reducing neurotoxicity and α-syn accumulation highlighting its potential in the treatment of PD.

Defective autophagy and increased apoptosis contribute toward the pathogenesis of FKRP-associated muscular dystrophies.

Fukutin-related protein (FKRP) is a glycosyltransferase involved in glycosylation of alpha-dystroglycan (α-DG). Mutations in FKRP are associated with muscular dystrophies (MD) ranging from limb-girdle LGMDR9 to Walker-Warburg Syndrome (WWS), a severe type of congenital MD. Although hypoglycosylation of α-DG is the main hallmark of this group of diseases, a full understanding of the underlying pathophysiology is still missing. Here, we investigated molecular mechanisms impaired by FKRP mutations in pluripotent stem (PS) cell-derived myotubes. FKRP-deficient myotubes show transcriptome alterations in genes involved in extracellular matrix receptor interactions, calcium signaling, PI3K-Akt pathway, and lysosomal function. Accordingly, using a panel of patient-specific LGMDR9 and WWS induced PS cell-derived myotubes, we found a significant reduction in the autophagy-lysosome pathway for both disease phenotypes. In addition, we show that WWS myotubes display decreased ERK1/2 activity and increased apoptosis, which were restored in gene edited myotubes. Our results suggest the autophagy-lysosome pathway and apoptosis may contribute to the FKRP-associated MD pathogenesis.

Overexpression of α-synuclein inhibits mitochondrial Ca2+ trafficking between the endoplasmic reticulum and mitochondria through MAMs by altering the GRP75-IP3R interaction.

Mitochondria-associated ER membranes (MAMs) are formed by close and specific components in the contact sites between the endoplasmic reticulum (ER) and mitochondria, which participate in several cell functions, including lipid metabolism, autophagy, and Ca2+ signaling. Particularly, the presence of α-synuclein (α-syn) in MAMs was previously demonstrated, indicating a physical interaction among some proteins in this region and a potential involvement in cell dysfunctions. MAMs alterations are associated with neurodegenerative diseases such as Parkinson's disease (PD) and contribute to the pathogenesis features. Here, we investigated the effects of α-syn on MAMs and Ca2+ transfer from the ER to mitochondria in WT- and A30P α-syn-overexpressing SH-SY5Y or HEK293 cells. We observed that α-syn potentiates the mitochondrial membrane potential (Δψm ) loss induced by rotenone, increases mitophagy and mitochondrial Ca2+ overload. Additionally, in α-syn-overexpressing cells, we found a reduction in ER-mitochondria contact sites through the impairment of the GRP75-IP3R interaction, however, with no alteration in VDAC1-GRP75 interaction. Consequently, after Ca2+ release from the ER, α-syn-overexpressing cells demonstrated a reduction in Ca2+ buffering by mitochondria, suggesting a deregulation in MAM activity. Taken together, our data highlight the importance of the α-syn/MAMs/Ca2+ axis that potentially affects cell functions in PD.

Pharmacological Modulators of Autophagy as a Potential Strategy for the Treatment of COVID-19.

The family of coronaviruses (CoVs) uses the autophagy machinery of host cells to promote their growth and replication; thus, this process stands out as a potential target to combat COVID-19. Considering the different roles of autophagy during viral infection, including SARS-CoV-2 infection, in this review, we discuss several clinically used drugs that have effects at different stages of autophagy. Among them, we mention (1) lysosomotropic agents, which can prevent CoVs infection by alkalinizing the acid pH in the endolysosomal system, such as chloroquine and hydroxychloroquine, azithromycin, artemisinins, two-pore channel modulators and imatinib; (2) protease inhibitors that can inhibit the proteolytic cleavage of the spike CoVs protein, which is necessary for viral entry into host cells, such as camostat mesylate, lopinavir, umifenovir and teicoplanin and (3) modulators of PI3K/AKT/mTOR signaling pathways, such as rapamycin, heparin, glucocorticoids, angiotensin-converting enzyme inhibitors (IECAs) and cannabidiol. Thus, this review aims to highlight and discuss autophagy-related drugs for COVID-19, from in vitro to in vivo studies. We identified specific compounds that may modulate autophagy and exhibit antiviral properties. We hope that research initiatives and efforts will identify novel or "off-label" drugs that can be used to effectively treat patients infected with SARS-CoV-2, reducing the risk of mortality.

Angiotensin II modulates the murine hematopoietic stem cell and progenitors cocultured with stromal S17 cells.

Although the existence of the renin-angiotensin system (RAS) in the bone marrow is clear, the exact role of this system in hematopoiesis has not yet been fully characterized. Here the direct role of angiotensin II (AngII) in hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/monocyte progenitors (GMPs), and megakaryocytes/erythroid progenitors (MEPs), using a system of coculture with stromal S17 cells. Flow cytometry analysis showed that AngII increases the percentage of HSC and GMP, while reducing CMP with no effect on MEP. According to these data, AngII increased the total number of mature Gr-1+ /Mac-1+ cells without changes in Terr119+ cells. AngII does not induce cell death in the population of LSK cells. In these populations, treatment with AngII decreases the expression of Ki67+ protein with no changes in the Notch1 expression, suggesting a role for AngII on the quiescence of immature cells. In addition, exposure to AngII from murine bone marrow cells increased the number of CFU-GM and BFU-E in a clonogenic assay. In conclusion, our data showed that AngII is involved in the regulation of hematopoiesis with a special role in HSC, suggesting that AngII should be evaluated in coculture systems, especially in cases that require the expansion of these cells in vitro, still a significant challenge for therapeutic applications in humans.

Cannabidiol induces autophagy via ERK1/2 activation in neural cells.

Autophagy is a lysosomal catabolic process essential to cell homeostasis and is related to the neuroprotection of the central nervous system. Cannabidiol (CBD) is a non-psychotropic phytocannabinoid present in Cannabis sativa. Many therapeutic actions have been linked to this compound, including autophagy activation. However, the precise underlying molecular mechanisms remain unclear, and the downstream functional significance of these actions has yet to be determined. Here, we investigated CBD-evoked effects on autophagy in human neuroblastoma SH-SY5Y and murine astrocyte cell lines. We found that CBD-induced autophagy was substantially reduced in the presence of CB1, CB2 and TRPV1 receptor antagonists, AM 251, AM 630 and capsazepine, respectively. This result strongly indicates that the activation of these receptors mediates the autophagic flux. Additionally, we demonstrated that CBD activates autophagy through ERK1/2 activation and AKT suppression. Interestingly, CBD-mediated autophagy activation is dependent on the autophagy initiator ULK1, but mTORC1 independent. Thus, it is plausible that a non-canonical pathway is involved. Our findings collectively provide evidence that CBD stimulates autophagy signal transduction via crosstalk between the ERK1/2 and AKT kinases, which represent putative regulators of cell proliferation and survival. Furthermore, our study sheds light on potential therapeutic cannabinoid targets that could be developed for treating neurodegenerative disorders.

α-Synuclein Overexpression Induces Lysosomal Dysfunction and Autophagy Impairment in Human Neuroblastoma SH-SY5Y.

Although the etiology of Parkinson's disease (PD) is multifactorial, it has been linked to abnormal accumulation of α-synuclein (α-syn) in dopaminergic neurons, which could lead to dysfunctions on intracellular organelles, with potential neurodegeneration. Patients with familial early-onset PD frequently present mutation in the α-syn gene (SNCA), which encodes mutant α-syn forms, such as A30P and A53T, which potentially regulate Ca2+ unbalance. Here we investigated the effects of overexpression of wild-type α-syn (WT) and the mutant forms A30P and A53T, on modulation of lysosomal Ca2+ stores and further autophagy activation. We found that in α-syn-overexpressing cells, there was a decrease in Ca2+ released from endoplasmic reticulum (ER) which is related to the increase in lysosomal Ca2+ release, coupled to lysosomal pH alkalization. Interestingly, α-syn-overexpressing cells showed lower LAMP1 levels, and a disruption of lysosomal morphology and distribution, affecting autophagy. Interestingly, all these effects were more evident with A53T mutant isoform when compared to A30P and WT α-syn types, indicating that the pathogenic phenotype for PD is potentially related to impairment of α-syn degradation. Taken together, these events directly impact PD-related dysfunctions, being considered possible molecular targets for neuroprotection.

Effects of ICI 182,780, an ERα and ERß antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells.

OBJECTIVE: To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERß) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. METHODS: Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. RESULTS: Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. CONCLUSION: The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.

Effective Synergy of Sorafenib and Nutrient Shortage in Inducing Melanoma Cell Death through Energy Stress.

Skin melanoma is one of the most aggressive and difficult-to-treat human malignancies, characterized by poor survival rates, thus requiring urgent novel therapeutic approaches. Although metabolic reprogramming has represented so far, a cancer hallmark, accumulating data indicate a high plasticity of cancer cells in modulating cellular metabolism to adapt to a heterogeneous and continuously changing microenvironment, suggesting a novel therapeutic approach for dietary manipulation in cancer therapy. To this aim, we exposed melanoma cells to combined nutrient-restriction/sorafenib. Results indicate that cell death was efficiently induced, with apoptosis representing the prominent feature. In contrast, autophagy was blocked in the final stage by this treatment, similarly to chloroquine, which also enhanced melanoma cell sensitization to combined treatment. Energy stress was evidenced by associated treatment with mitochondrial dysfunction and glycolysis impairment, suggesting metabolic stress determining melanoma cell death. A reduction of tumor growth after cycles of intermittent fasting together with sorafenib treatment was also observed in vivo, reinforcing that the nutrient shortage can potentiate anti-melanoma therapy. Our findings showed that the restriction of nutrients by intermittent fasting potentiates the effects of sorafenib due to the modulation of cellular metabolism, suggesting that it is possible to harness the energy of cancer cells for the treatment of melanoma.

Cyclopalladated compounds containing 2,6-lutidine: Synthesis, spectral and biological studies.

Bridge splitting reactions between [Pd(C2,N-dmba)(µ-X)]2 (dmba = N,N-dimethylbenzylamine; X = Cl, I, N3, NCO) and 2,6-lutidine (lut) in the 1:2 molar ratio at room temperature afforded cyclopalladated compounds of general formulae [Pd(C2,N-dmba)(X)(lut)] {X = Cl- (1), I-(2), NNN-(3), NCO-(4)}, which were characterized by elemental analyses and infrared (IR), 1H NMR spectroscopy. The molecular structures of all synthesized palladacycles have been solved by single-crystal X-ray crystallography. The cytotoxicity of the cyclopalladated compounds has been evaluated against a panel of murine {mammary carcinoma (4T1) and melanoma (B16F10-Nex2)} and human {melanoma (A2058, SK-MEL-110 and SK-MEL-5) tumor cell lines. All complexes were about 10 to 100-fold more active than cisplatin, depending on the tested tumor cell line. For comparison purposes, the cytotoxic effects of 1-4 towards human lung fibroblasts (MRC-5) have also been tested. The late apoptosis-inducing properties of 1-4 compounds in SK-MEL-5 cells were verified 24 h incubation using annexin V-Fluorescein isothiocyanate (FITC)/propidium iodide (PI). The binding properties of the model compound 1 on human serum albumin (HSA) and calf thymus DNA (ct-DNA) have been studied using circular dichroism and fluorescence spectroscopy. Docking simulations have been carried out to gain more information about the interaction of the palladacycle and HSA. The ability of compounds 1-4 to inhibit the activity of cathepsin B and L has also been investigated in this work.

Effects of ICI 182, 780, an ERα and ERß antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells / Efeitos do antagonista seletivo do REα e do REß ICI 182, 780 e do agonista de GPER G-1 no processo de autofagia em células de câncer de mama

ABSTRACT Objective To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. Methods Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. Results Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. Conclusion The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.

RESUMO Objetivo Avaliar o efeito dos compostos ICI 182,780 (fulvestranto), um antagonista seletivo dos receptores de estrógeno alfa/beta (REα/REβ), e do G-1, um agonista seletivo de receptores de estrógeno acoplados a proteínas-G (GPER), na possível indução de autofagia em linhagens de câncer de mama MCF-7 e SKBr3, bem como o efeito de G-1 na viabilidade celular. Métodos A viabilidade celular de células MCF-7 e SKBr3 foi avaliada pelo ensaio com MTT. Para investigar a indução da autofagia, células MCF-7 foram transfectadas com GFP-LC3, um marcador de autofagossomos, e analisadas por microscopia de fluorescência em tempo real. As células MCF-7 e SKBr3 foram incubadas com o indicador de compartimentos ácidos laranja de acridina e analisadas por citometria de fluxo como indicativo para autofagia. Resultados Em células MCF-7, o ICI 182,780 e rapamicina após 48 horas levaram à diminuição da viabilidade celular, enquanto o G-1 não alterou a viabilidade no mesmo período de tratamento. Nem o ICI 182,780 e nem o G-1 induziram aumento na pontuação de GFP-LC3 em células MCF-7 até 4 horas. Já os ensaios de citometria de fluxo demonstraram que ICI 182,780 levou ao aumento de compartimentos ácidos em células MCF-7. O G-1 não aumentou estes parâmetros em ambas as linhagens. Por outro lado, ICI 182,780 e G-1 não induziram à redução da viabilidade em células SKBr3 e nem à formação de compartimentos ácidos, como etapa final do processo autofágico. Conclusão O aumento de compartimentos ácidos pelo ICI 182,780 em células de câncer de mama positivas para receptores de estrógeno parece estar associado com seu efeito inibidor de receptores de estrógeno, mas sem o envolvimento de GPER. A compreensão desses mecanismos pode direcionar estudos sobre o envolvimento dos receptores nos processos celulares de resistência do câncer de mama.

Medicinal properties of Angelica archangelica root extract: Cytotoxicity in breast cancer cells and its protective effects against in vivo tumor development.

OBJECTIVE: Although Angelica archangelica is a medicinal and aromatic plant with a long history of use for both medicinal and food purposes, there are no studies regarding the antineoplastic activity of its root. This study aimed to evaluate the cytotoxicity and antitumor effects of the crude extract of A. archangelica root (CEAA) on breast cancer. METHODS: The cytotoxicity of CEAA against breast adenocarcinoma cells (4T1 and MCF-7) was evaluated by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Morphological and biochemical changes were detected by Hoechst 33342/propidium iodide (PI) and annexin V/PI staining. Cytosolic calcium mobilization was evaluated in cells staining with FURA-4NW. Immunoblotting was used to determine the effect of CEAA on anti- and pro-apoptotic proteins (Bcl-2 and Bax, respectively). The 4T1 cell-challenged mice were used for in vivo assay. RESULTS: Using ultra-high-performance liquid chromatography-mass spectrometry analysis, angelicin, a constituent of the roots and leaves of A. archangelica, was found to be the major constituent of the CEAA evaluated in this study (73 µg/mL). The CEAA was cytotoxic for both breast cancer cell lines studied but not for human fibroblasts. Treatment of 4T1 cells with the CEAA increased Bax protein levels accompanied by decreased Bcl-2 expression, in the presence of cleaved caspase-3 and cytosolic calcium mobilization, suggesting mitochondrial involvement in breast cancer cell death induced by the CEAA in this cell line. No changes on the Bcl-2/Bax ratio were observed in CEAA-treated MCF7 cells. Gavage administration of the CEAA (500 mg/kg) to 4T1 cell-challenged mice significantly decreased tumor growth when compared with untreated animals. CONCLUSION: Altogether, our data show the antitumor potential of the CEAA against breast cancer cells in vitro and in vivo. Further research is necessary to better elucidate the pharmacological application of the CEAA in breast cancer therapy.

Autophagy and intermittent fasting: the connection for cancer therapy?

Cancer is a leading cause of death worldwide, and its incidence is continually increasing. Although anticancer therapy has improved significantly, it still has limited efficacy for tumor eradication and is highly toxic to healthy cells. Thus, novel therapeutic strategies to improve chemotherapy, radiotherapy and targeted therapy are an important goal in cancer research. Macroautophagy (herein referred to as autophagy) is a conserved lysosomal degradation pathway for the intracellular recycling of macromolecules and clearance of damaged organelles and misfolded proteins to ensure cellular homeostasis. Dysfunctional autophagy contributes to many diseases, including cancer. Autophagy can suppress or promote tumors depending on the developmental stage and tumor type, and modulating autophagy for cancer treatment is an interesting therapeutic approach currently under intense investigation. Nutritional restriction is a promising protocol to modulate autophagy and enhance the efficacy of anticancer therapies while protecting normal cells. Here, the description and role of autophagy in tumorigenesis will be summarized. Moreover, the possibility of using fasting as an adjuvant therapy for cancer treatment, as well as the molecular mechanisms underlying this approach, will be presented.

Lithium, a classic drug in psychiatry, improves nilotinib-mediated antileukemic effects.

Although Tyrosine kinase inhibitors (TKIs) that target Bcr-Abl play a key role in Chronic Myeloid Leukemia (CML) therapy, they do not eradicate CML-initiating cells, which lead to the emergence of drug resistance. Here we used the lithium, a GSK-3 inhibitor, to attempt to potentiate the effects of nilotinib against leukemia cells. For this purpose, a K562 leukemia cell line and bone marrow cells from untreated Chronic Myeloid Leukemia (CML) patients, prior to any exposure to TKIs, were used as a model. Our results demonstrated that the combination of lithium + nilotinib (L + N) induced K562-cell death and cleaved caspase-3 when compared to lithium or nilotinib alone, accompanied by GSK-3ß phosphorylation and Bcr-Abl oncoprotein levels reduction. Interestingly, these events were related to autophagy induction, expressed by increased LC3II protein levels in the group treated with L + N. Furthermore, the clonogenic capacity of progenitor cells from CML patients was drastically reduced by L + N, as well as lithium and nilotinib when used separately. The number of cell aggregates (clusters), were increased by all treatments (L + N, lithium, and nilotinib). This pioneering research has demonstrated that lithium might be of therapeutic value when targeting Bcr-Abl cells with nilotinib because it triggers cell death in addition to exerting classical antiproliferative effects, opening new perspectives for novel target and therapeutic approaches to eradicate CML.

Overexpression of α-synuclein in an astrocyte cell line promotes autophagy inhibition and apoptosis.

α-Synuclein is the major component of neuronal cytoplasmic aggregates called Lewy bodies, the main pathological hallmark of Parkinson disease. Although neurons are the predominant cells expressing α-synuclein in the brain, recent studies have demonstrated that primary astrocytes in culture also express α-synuclein and regulate α-synuclein trafficking. Astrocytes have a neuroprotective role in several detrimental brain conditions; we therefore analyzed the effects of the overexpression of wild-type α-synuclein and its A30P and A53T mutants on autophagy and apoptosis. We observed that in immortalized astrocyte cell lines, overexpression of α-synuclein proteins promotes the decrease of LC3-II and the increase of p62 protein levels, suggesting the inhibition of autophagy. When these cells were treated with rotenone, there was a loss of mitochondrial membrane potential, especially in cells expressing mutant α-synuclein. The level of this decrease was related to the toxicity of the mutants because they show a more intense and sustained effect. The decrease in autophagy and the mitochondrial changes in conjunction with parkin expression levels may sensitize astrocytes to apoptosis.