Nanozyme-Based Lateral Flow Immunoassay (LFIA) for Extracellular Vesicle Detection.

Extracellular vesicles (EVs) are biological nanoparticles of great interest as novel sources of biomarkers and as drug delivery systems for personalized therapies. The research in the field and clinical applications require rapid quantification. In this study, we have developed a novel lateral flow immunoassay (LFIA) system based on Fe3O4 nanozymes for extracellular vesicle (EV) detection. Iron oxide superparamagnetic nanoparticles (Fe3O4 MNPs) have been reported as peroxidase-like mimetic systems and competent colorimetric labels. The peroxidase-like capabilities of MNPs coated with fatty acids of different chain lengths (oleic acid, myristic acid, and lauric acid) were evaluated in solution with H2O2 and 3,3,5,5-tetramethylbenzidine (TMB) as well as on strips by biotin-neutravidin affinity assay. As a result, MNPs coated with oleic acid were applied as colorimetric labels and applied to detect plasma-derived EVs in LFIAs via their nanozyme effects. The visual signals of test lines were significantly enhanced, and the limit of detection (LOD) was reduced from 5.73 × 107 EVs/µL to 2.49 × 107 EVs/µL. Our work demonstrated the potential of these MNPs as reporter labels and as nanozyme probes for the development of a simple tool to detect EVs, which have proven to be useful biomarkers in a wide variety of diseases.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor ß secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

Mechanisms involved in the pro-apoptotic effect of melatonin in cancer cells.

It is well established that melatonin exerts antitumoral effects in many cancer types, mostly decreasing cell proliferation at low concentrations. On the other hand, induction of apoptosis by melatonin has been described in the last few years in some particular cancer types. The cytotoxic effect occurs after its administration at high concentrations, and the molecular pathways involved have been only partially determined. Moreover, a synergistic effect has been found in several cancer types when it is administered in combination with chemotherapeutic agents. In the present review, we will summarize published work on the pro-apoptotic effect of melatonin in cancer cells and the reported mechanisms involved in such action. We will also construct a hypothesis on how different cell signaling pathways may relate each other on account for such effect.

Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.

Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.

Cooperative action of JNK and AKT/mTOR in 1-methyl-4-phenylpyridinium-induced autophagy of neuronal PC12 cells.

Parkinson's disease has been widely related to both apoptosis and oxidative stress. Many publications relate the loss of mitochondrial potential to an apoptosis-mediated cell death in different in vivo and in vitro models of this pathology. The present study used the dopaminegic specific neurotoxin 1-methyl-4-phenylpyridinium (MPP(+) ) on neuron-like PC12 cells, which is a well-accepted model of Parkinson's disease. Results showed an early increase in oxidants, which drives the modulation of c-Jun N-terminal kinase (JNK) and AKT/mammalian target of rapamycin (mTOR) pathways, mimicking peroxide treatment. However, the cell death found in neuronal PC12 cells treated with MPP(+) was not a caspase-associated apoptosis. Electron microscopic images illustrated autophagic cell death, which was confirmed by a Beclin-1 and ATG expression increase, accumulation of acidic vesicles, and rescue by an autophagy inhibitor. In conclusion, the boost in oxidants from MPP(+) treatment in neuronal PC12 is modulating both survival (AKT/mTOR) and death (JNK) pathways, which are the perpetrators of an autophagic cell death.

Intracellular redox state as determinant for melatonin antiproliferative vs cytotoxic effects in cancer cells.

Melatonin is an endogenous indolamine, classically known as a light/dark regulator. Besides classical functions, melatonin has also showed to have a wide range of antitumoral effects in numerous cancer experimental models. However, no definite mechanism has been described to explain the whole range of antineoplasic effects. Here we describe a dual effect of melatonin on intracellular redox state in relation to its antiproliferative vs cytotoxic actions in cancer cells. Thus, inhibition of proliferation correlates with a decrease on intracellular reactive oxygen species (ROS) and increase of antioxidant defences (antioxidant enzymes and intracellular gluthation,GSH levels), while induction of cell death correlates with an increase on intracellular ROS and decrease of antioxidant defences. Moreover, cell death can be prevented by other well-known antioxidants or can be increased by hydrogen peroxide. Thus, tumour cell fate will depend on the ability of melatonin to induce either an antioxidant environment--related to the antiproliferative effect or a prooxidant environment related to the cytotoxic effect.

Regulation of the expression of death receptors and their ligands by melatonin in haematological cancer cell lines and in leukaemia cells from patients.

Incorporation of new therapeutic agents remains as a major challenge for treatment of patients with malignant haematological disorders. Melatonin is an indolamine without relevant side effects. It has been shown previously to exhibit synergism with several chemotherapeutic drugs in Ewing sarcoma cells by potentiating the extrinsic pathway of apoptosis. It also sensitizes human glioma cells against TRAIL by increasing DR5 expression. Here, we report the induction of cell death by melatonin in several human malignant haematological cell lines through the activation of the extrinsic pathway of apoptosis. Such activation was mediated by the increase in the expression of the death receptors Fas, DR4 and DR5 and their ligands Fas L and TRAIL, with a remarkable rise in the expression of Fas and Fas L. The cytotoxic effect and the increase in Fas and Fas L were dependent on Akt activation. Results were corroborated in blasts from bone marrow and peripheral blood of acute myeloid leukaemia patients, where melatonin induced cell death and increased both Fas and Fas L expressions. We conclude that melatonin may be considered as a potential antileukaemic agent and its therapeutic use, either alone or in combination with current chemotherapeutic drugs, should be taken into consideration for further research.

Synergistic antitumor effect of melatonin with several chemotherapeutic drugs on human Ewing sarcoma cancer cells: potentiation of the extrinsic apoptotic pathway.

Ewing sarcoma, the second most frequent bone cancer type, affects mainly adolescents, who have a survival of 50% 5 yr after diagnosis. Current treatments include a combination of surgery, radiotherapy and chemotherapy, which present potential serious side effects. Melatonin, a natural molecule without relevant side effects, has been previously shown to induce cytotoxicity in SK-N-MC cells, a Ewing sarcoma cell line. Here, we found that there is a synergy in the antitumor effect when melatonin (50 mum-1 mm) is combined with vincristine at the concentration of 5-10 nm or with ifosfamide at the range of 100 mum-1 mm. This synergism is due to the potentiation of cell death, particularly to the potentiation of apoptosis, i.e., mainly the extrinsic apoptotic pathway. There is a significant increase in the activation of caspase-3, -8, -9 and Bid when melatonin is combined with vincristine or ifosfamide compared to the individual treatments. Finally, there is also a potentiation of the early free radical production, likely dependent on the extrinsic apoptosis pathway activation, when the drugs are combined with melatonin. Other proteins which are related to this pathway including mitogen-activated protein kinase or protein kinase B/Akt are not involved in apoptosis induced by these agents separately or when combined. The results shown here together with the facts that: (i) no relevant side effects have been reported for melatonin and (ii) melatonin has a cytoprotective effect on noncancer cells, opens the door for a new approach in the treatment of the Ewing sarcoma family of tumors.

Melatonin sensitizes human malignant glioma cells against TRAIL-induced cell death.

Despite the common expression of death receptors, many types of cancer including gliomas are resistant to the death receptor ligand (TRAIL). Melatonin antitumoral actions have been extensively described, including oncostatic properties on several tumor types and improvement of chemotherapeutic regimens. Here, we found that melatonin effectively increase cell sensitivity to TRAIL-induced cell apoptosis in A172 and U87 human glioma cells. The effect seems to be related to a modulation of PKC activity which in turns decreases Akt activation leading to an increase in death receptor 5 (DR5) levels and a decrease in the antiapoptotic proteins survivin and bcl-2 levels.

Intracellular signaling pathways involved in post-mitotic dopaminergic PC12 cell death induced by 6-hydroxydopamine.

Oxidative stress has been shown to mediate neuron damage in Parkinson's disease (PD). In the present report, we intend to clarify the intracellular pathways mediating dopaminergic neuron death after oxidative stress production using post-mitotic PC12 cells treated with the neurotoxin 6-hydroxydopamine (6-OHDA). The use of post-mitotic cells is crucial, because one of the suggested intracellular pathways implicated in neuron death relates to the re-entry of neurons (post-mitotic cells) in the cell cycle. We find that 6-OHDA sequentially increases intracellular oxidants, functional cell damage and caspase-3 activation, leading to cell death after 12 h of incubation. Prevention of cell damage by different antioxidants supports the implication of oxidative stress in the observed neurotoxicity. Oxidative stress-dependent phosphorylation of the MAPK JNK and oxidative stress-independent PKB/Akt dephosphorylation are involved in 6-OHDA neurotoxicity. Decrease in p21(WAF1/CIP1) and cyclin-D1 expression, disappearance of the non-phosphorylated band of retinoblastoma protein (pRb), and expression of proliferating cell nuclear antigen, not present in PC12 post-mitotic cells, suggest a re-entry of differentiated cells into cell cycle. Our results indicate that such a re-entry is mediated by oxidative stress and is involved in 6-OHDA-induced cell death. We conclude that at least three intracellular pathways are involved in 6-OHDA-induced cell death in differentiated PC12 cells: JNK activation, cell cycle progression (both oxidative stress-dependent), and Akt dephosphorylation (not related to the increase of oxidants); the three pathways are necessary for the cells to die, since blocking one of them is sufficient to keep the cells alive.

Involvement of protein kinase C in melatonin's oncostatic effect in C6 glioma cells.

Classical anticancer therapies often are ineffective in patients with malignant glioma who have a survival of <1 year. Our previous studies showed a potent inhibitory effect of melatonin on glioma cell proliferation. This effect seems to be mediated by the well-known antioxidant properties of this molecule and the negative regulation of some intracellular effectors, such as the kinase Akt or the transcription factor nuclear factor (NF)-kappaB. Finally, protein kinase C (PKC) also seems to be implicated in this effect although the intracellular pathways involved have not been elucidated. In this study, we analyzed the role of PKC in the regulation by melatonin of intracellular effectors leading to inhibition of cell proliferation. Activation of PKC by incubation with triphorbol ester acetate (TPA) blocks the inhibitory effect of melatonin on Akt and NF-kappaB activity. Moreover, incubation with melatonin induces a decrease in p21 expression in these cells that is partially blocked by co-incubation with TPA. Taken together, these results suggest that melatonin's oncostatic effect on glioma cells is mediated, at least in part, by the inhibition of PKC activity which, in turn, results in Akt and NF-kappaB activity inhibition and modulation of cell cycle-related gene expression.

Signaling pathways involved in antioxidant control of glioma cell proliferation.

Tumor cells are able to survive and proliferate despite the higher-than-average level of reactive oxygen species (ROS) they exhibit. This is generally taken as a clue as to the implications of ROS in cell proliferation. In fact many mitogenic intracellular signaling pathways could be redox regulated, more particularly those involving tyrosine kinase receptors (RTK). In the present work we use N-acetylcysteine (NAC)-a well-known antioxidant molecule-to study the implications of cellular redox state on rat C6 glioma cell proliferation. NAC is shown to decrease glioma cell proliferation, inducing a cell cycle arrest in the G(0)/G(1) phase and markedly up-regulating p21 expression. A rapid, and glutathione-independent, decrease in intracellular oxidants was observed as well. NAC also lowers Akt activity, extracellular signal-regulated kinase 1/2, and the redox-sensitive transcription factor NF-kappaB, all of which are ROS related and seem to be in close connection with cell proliferation. NAC effects apparently relate to protein kinase C (PKC) activity because 100 nM TPA-a PKC activator-induces a partial blockage of the NAC antiproliferative effect. Bringing our results together, it seems that intracellular reduction of oxidants in C6 glioma cells can induce inhibition of cell proliferation by modulating RTK-related intracellular signaling pathways.

Melatonin prevents glutamate-induced oxytosis in the HT22 mouse hippocampal cell line through an antioxidant effect specifically targeting mitochondria.

The pineal hormone melatonin has neuroprotective effects in a large number of models of neurodegeneration. Melatonin crosses the blood-brain barrier, shows a decrease in its nocturnal peaks in blood with age that has been associated with the development of neurodegenerative disorders, and has been shown to be harmless at high concentrations. These properties make melatonin a potential therapeutic agent against neurodegenerative disorders but the pathways involved in such neuroprotective effects remain unknown. In the present report we study the intracellular pathways implicated in the complete neuroprotection provided by melatonin against glutamate-induced oxytosis in the HT22 mouse hippocampal cell line. Our results strongly suggest that melatonin prevents oxytosis through a direct antioxidant effect specifically targeted at the mitochondria. Firstly, none of the described transducers of melatonin signalling seems to be implicated in the neuroprotection provided by this indole. Secondly, melatonin does not prevent cytosolic GSH depletion-dependent increase in reactive oxygen species (ROS), but it totally prevents mitochondrial ROS production despite the fact that the latter is much higher than the former. And finally, there is a high correlation between the concentration at which melatonin and closely related indoles exert a direct antioxidant effect in vitro and a neuroprotective effect against glutamate-induced oxytosis.

Melatonin induces apoptosis in human neuroblastoma cancer cells.

Low concentrations (nanomolar) of melatonin had been previously shown to inhibit cell proliferation in several cancer cell lines as well as in experimental animal models. Additionally, cell growth inhibition and differentiation of prostate cancer cell lines by high concentrations (micromolar to millimolar) of melatonin have been recently reported. In the present paper, we show the induction of apoptosis by high doses of melatonin in the human neuroblastoma cell line SK-N-MC. We found accumulation of cells in the G2/M cell cycle phase and induction of cellular death, measured as lactate dehydrogenase (LDH) released into the culture medium, under millimolar concentration of melatonin. Apoptosis was evaluated using 4,6-diamidino-2-phenylindole staining, DNA gel electrophoresis, electron microscopy, and annexin V binding. Apoptosis progressed through the classical pathway, which involves caspase-3 activation. Cell death was dose and time-dependent; the lowest effective concentration of melatonin was 100 microm. Treatment with 1 mm melatonin for 6 days induced cell death in 75% of the cells. This novel finding shows that a nontoxic natural indoleamine may be potential therapy for some types of human neuroblastomas.

Tryptamine induces cell death with ultrastructural features of autophagy in neurons and glia: Possible relevance for neurodegenerative disorders.

Tryptamine derivatives are a family of biogenic amines that have been suggested to be modulators of brain function at physiological concentrations. However, pharmacological concentrations of these amines display amphetamine-like properties, and they seem to play a role in brain disorders. Amphetamines induce autophagy in nerve cells, and this type of cell death has also been involved in neurodegenerative diseases. In the present work, we clearly demonstrate for the very first time that high concentrations of tryptamine (0.1-1 mM) induce autophagy in HT22 and SK-N-SH nerve cell lines and in primary cultures of astrocytes, glial cells being less sensitive than neurons. Ultrastructural cell morphology shows all of the typical hallmarks of autophagy. There is no nuclear chromatin condensation, endoplasmic reticulum and mitochondria are swollen, and a great number of double-membraned autophagosomes and residual bodies can be shown in the cytoplasm. Autophagosomes and residual bodies contain mitochondria, membranes, and vesicles and remain unabridged until the cell membrane is disrupted and the cell dies. The same results have been found when cells were incubated with high concentrations of 5-methoxytryptamine (0.1-1 mM). Our results establish a possible link between the role of tryptamine derivatives in brain disorders and the presence of autophagic cell death in these kinds of disorders.

Intracellular signaling pathways involved in the cell growth inhibition of glioma cells by melatonin.

Melatonin is an indolamine mostly produced in the pineal gland, soluble in water, and highly lipophilic, which allows it to readily cross the blood-brain barrier. Melatonin possesses antioxidant properties and its long-term administration in rodents has not been found to cause noteworthy side effects. In the present work, we found that millimolar concentrations of this indolamine reduced cell growth of C6 glioma cells by 70% after 72 hours of treatment, inhibiting cell progression from G(1) to S phase of the cell cycle. Intraperitoneal administration of 15 mg/kg body weight of melatonin to rats previously injected in the flank with C6 glioma cells reduces tumor growth by 50% 2 weeks after the implant. Inhibition of cell growth does not depend on melatonin membrane receptor activation whereas it seemingly relates to the reduction of intracellular basal free radical levels by 30%. Increase of basal redox state of the cells and constitutive activation of tyrosine kinase receptor [receptor tyrosine kinase (RTK)] pathways, including the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Akt and protein kinase C (PKC) signaling pathways, contribute to the progression of the gliomas leading to the constitutive activation of the redox-dependent survival transcription factor nuclear factor kappaB (NF-kappaB). The antioxidant effect of melatonin in C6 cells is associated to inhibition of NF-kappaB and Akt, but not of ERK1/2. The antiproliferative effect of the indolamine on these cells is partially abolished when coincubated with the PKC activator 12-O-tetradecanoylphorbol-13-acetate, thus indicating that the ability of melatonin to change cellular redox state may be inactivating the pathway RTK/PKC/Akt/NF-kappaB.

Standard curve for housekeeping and target genes: specific criteria for selection of loading control in Northern blot analysis.

The election of the correct loading control in Northern blot normalization is something essential to obtain valid results. Housekeeping genes are widely used as loading control and the assumption is made that they counteract load differences between samples. We have found, however, that uneven sample load is capable to alter the results despite normalization, considering no influence of the experimental conditions on housekeeping gene regulation takes place. Normalization ratio (transcript of interest/housekeeping gene) is determined as the pattern of variation in the ratio between densitometric signals of transcripts--both target and control--and the amount of total RNA. The fact that this relationship is specific for each transcript means different ratios will exist depending on the chosen control gene. Actually, loading differences of only 2 microg may induce a 2.5-fold difference between normalized ratios, depending on the housekeeping gene selected for normalization. In order to select the appropriate loading control, it becomes essential to establish a standard curve for each transcript of interest and several housekeeping. Only the one yielding a constant ratio of normalization along the total RNA range used is to be taken into consideration.

Intracellular redox state regulation by parthenolide.

In the present paper, we report a strong intracellular antioxidant activity of the sesquiterpene lactone parthenolide in the hippocampal HT22 cells. This effect is mediated by an increase of total glutathione at both, low (5 microM) and high (10 microM), concentrations. Parthenolide also increases the activation of the antioxidant/electrophile response element. This effect is the possible mediator of glutathione increase, since the limiting enzyme on its synthesis possesses this response element on its promoter. Finally, we demonstrate that its antioxidant properties do not mediate its antiproliferative effect nor its inhibition of NF-kappaB.