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OBJECTIVE: Short-chain fatty acids (SCFAs) are microbial derived metabolites, which have multiple beneficial properties. The amount of SCFAs depends on several factors, such as age, diet (mainly intake of dietary fiber), and overall health condition. The normal proportion between SCFAs is 3:1:1 for acetate, proprionate and butyrate, respectively. In colorectal cancer (CRC) patients, microbiota alterations have been shown. Consequently, metabolome within the gut might change to a large extent. Therefore, the aim of this study was to analyse the content of SCFAs and the proportion between SCFAs in the stool obtained from CRC patients in preoperative period. PATIENTS AND METHODS: This study included 15 patients with CRC in preoperative period. The stool samples were taken and stored at -80°C in the Fahrenheit Biobank BBMRI.pl, Medical University of Gdansk, Poland. The analysis of SCFAs from stool samples was conducted by means of gas chromatography. RESULTS: This study included mainly males (66.67%, n=10). In all patients, there was abnormal proportion between SCFAs. The extremely higher concentration of butyrate was noted in 2 samples (13.33%) compared to the rest of patients. However, based on normal proportion between SCFAs, the results <1 for butyrate were noted in 93.33% of patients. CONCLUSIONS: SCFAs pool is altered in CRC patients, among others characterized by low level of butyrate. It should be considered to administer butyrate supplementation to CRC patients especially prior to surgery to support an appropriate preparation to this treatment. [ABSTRACT FROM AUTHOR]

Resveratrol is a naturally occurring polyphenolic compound present in many plant species and wine. It possesses a wide range of beneficial biological properties including anticancer activity. Resveratrol has been demonstrated to induce both autophagy and apoptosis in several human cancer cell lines. The aim of this study was to investigate whether resveratrol modulates autophagy and apoptosis in MOLT-4 human lymphoblastic leukemia and HL-60 human promyelocytic leukemia cells. Cell viability was evaluated by the neutral red uptake assay. Cell cycle distribution, phosphatidylserine externalization, caspase-3 activation, changes of the mitochondrial membrane potential, intracellular production of reactive oxygen species were evaluated by flow cytometry. LC3-I to LC3-II conversion was examined based on Western blotting and immunofluorescence analyses. The level of p62/SQSTM1 protein and PARP1 cleavage were analyzed by Western blotting. The DNA degradation was assessed by gel electrophoresis. We found that resveratrol is able to modulate autophagy in MOLT-4 and HL-60 cells, as evidenced by the detection of an increased level of LC3-II and p62/SQSTM1 proteins. Moreover, resveratrol induced apoptosis in both cell lines which was associated with phosphatidylserine externalization, disruption of the mitochondrial membrane potential, caspase-3 activation, internucleosomal DNA fragmentation, PARP1 cleavage, chromatin condensation, and fragmentation of cell nuclei. The present study provides evidence that resveratrol can act as an autophagy modulator as well as an apoptosis inducer in MOLT-4 and HL-60 human leukemia cells. Our findings imply that resveratrol can be a promising chemotherapeutic agent in the treatment of leukemia. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia is characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells. Despite recent advances in the treatment of this disease, the prognosis and overall long-term survival for patients remain poor, which drives the search for new chemotherapeutics and treatment strategies. Piceatannol, a polyphenolic compound present in grapes and wine, appears to be a promising chemotherapeutic agent in the treatment of leukemia. The aim of the present study was to examine whether piceatannol induces autophagy and/or apoptosis in HL-60 human acute myeloid leukemia cells and whether HL-60 cells are able to acquire resistance to piceatannol toxicity. We found that piceatannol at the IC 90 concentration of 14 µM did not induce autophagy in HL-60 cells. However, it induced caspase-dependent apoptosis characterized by phosphatidylserine externalization, disruption of the mitochondrial membrane potential, caspase-3 activation, internucleosomal DNA fragmentation, PARP1 cleavage, chromatin condensation, and fragmentation of cell nuclei. Our findings also imply that HL-60 cells are able to acquire resistance to piceatannol toxicity via mechanisms related to MRP1 activity. Our results suggest that the use of piceatannol as a potential chemotherapeutic agent may be associated with the risk of multidrug resistance, warranting its use in combination with other chemotherapeutic agents.

BMAA (ß-N-methylamino-L-alanine) was originally found in the seeds of cycad Cycas micronesica in the 1960s. Some years later it was discovered that the amino acid is genuinely produced by endosymbiotic cyanobacteria. Further research has proven the neurotoxic activity of BMAA, leading to neurodegenerative disease diagnosed as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC). The aim of the present work was to examine the occurrence of BMAA in samples from Polish waterbodies. Both, the field cyanobacterial samples and the isolated cyanobacterial strains were analyzed. Also mussel and fish samples were checked for the BMAA accumulation. The additional goal was to assess the biological activity of BMAA in in vivo and in vitro assays. In waters of Northern Poland, BMAA was detected in cyanobacteria from Synechococcales, Oscillatoriales and Nostocales orders. The free and protein-bound forms of BMAA were detected in 9 and 4 (of 37) environmental samples, respectively. Both forms of BMAA were also identified in 2 out of 21 cyanobacterial strains isolated from Polish waterbodies. Our analyses of cyanobacterial material did not confirm the presence of soluble protein-bound form of BMAA. The amino acid was detected neither in the tissues of fish nor in the mussels. Biological activity of BMAA was tested with the application of hippocampal neural cell line HT22 and crustaceans: Thamnocephalus platyurus, Artemia franciscana and Daphnia magna. Among them, only D. magna assay revealed toxic effects of BMAA. The results of our research did not demonstrate the widespread production of BMAA by cyanobacteria from Northern Poland waters., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The role of autophagy is to degrade damaged or unnecessary cellular structures. Both in vivo and in vitro studies suggest a dual role of autophagy in cancer-it may promote the development of neoplasms, but it may also play a tumor protective function. The mechanism of autophagy depends on the genetic context, tumor stage and type, tumor microenvironment, or clinical therapy used. Autophagy also plays an important role in cell death as well as in the induction of chemoresistance of cancer cells. The following review describes the extensive autophagic cell death in relation to dietary polyphenols and cancer disease. The review documents increasing use of polyphenolic compounds in cancer prevention, or as agents supporting oncological treatment. Polyphenols are organic chemicals that exhibit antioxidant, anti-inflammatory, anti-angiogenic, and immunomodulating properties, and can also initiate the process of apoptosis. In addition, polyphenols reduce oxidative stress and protect against reactive oxygen species. This review presents in vitro and in vivo studies in animal models with the use of polyphenolic compounds such as epigallocatechin-3-gallate (EGCG), oleuropein, punicalgin, apigenin, resveratrol, pterostilbene, or curcumin and their importance in the modulation of autophagy-induced death of cancer cells.

A new series of N-(aryl/heteroaryl)-2-chlorobenzenesulfonamide derivatives 4-21 have been synthesized, and evaluated at the National Cancer Institute (USA) for their in vitro activities against a panel of 60 different human cancer cell lines. Among them, compounds 16, 20 and 21 exhibited remarkable cytotoxic activity against numerous human cancer cell lines. We found that sulfonamide derivative 21 appeared to be more selective than compounds 16 and 20. In comparison to compounds 16 and 20 it showed higher cytotoxic activity against A549 non-small cell lung adenocarcinoma and HCT-116 colon carcinoma cells and was less toxic to HEK-293 human embryonic kidney cells and HaCaT immortalized human keratinocytes. Treatment of A549 and HCT-116 cells with compound 21 resulted in the G0/G1-cell cycle arrest with a concomitant increase in p53 and p21 protein levels. Moreover, compound 21 led to ATP depletion and disruption of the mitochondrial membrane potential in both studied cell lines. Our results suggest that 2,4-dichloro-N-(quinolin-8-yl and/or 1H-indazol-7-yl)benzenesulfonamides serve as novel promising anticancer agents., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Piceatannol, a polyphenolic compound present in grapes and wine, has been reported to exhibit anticancer properties. Recently, it has been demonstrated to exert antiproliferative and proapoptotic effects in various human cancer types. The aim of our study was to investigate whether piceatannol induces autophagy and apoptosis in MOLT-4 human leukemia cells. Our results revealed that piceatannol activated autophagy in MOLT-4 cells, as evidenced by the detection of an increased level of LC3-II protein and a concomitant decrease in p62/SQSTM1 protein level. Moreover, piceatannol induced apoptosis in MOLT-4 cells which was accompanied by phosphatidylserine externalization, caspase-3 activation, disruption of mitochondrial membrane potential, internucleosomal DNA fragmentation, PARP1 cleavage, chromatin condensation, and fragmentation of cell nuclei. However, the toxic effects exerted by piceatannol in MOLT4 cells diminished after longer periods of exposure to the compound. Our findings imply that MOLT-4 cells may acquire resistance to piceatannol toxicity, which may result from the induction of efflux transporters such as P-glycoprotein. The present study provides new data showing that the use of piceatannol as a potential chemotherapeutic agent in the treatment of leukemia may be associated with the risk of multidrug resistance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of this study was to examine the effects of 5‑fluorouracil (5‑FU), anti‑epidermal growth factor receptor (EGFR) antibody and aspirin (ASA) on the characteristics of two CRC cell lines, HCT116 and HT29, maintained in a spherical culture system. We observed that the morphology of both the HCT116 and HT29 cell‑derived spheres was significantly impaired and the size of the colonospheres was markedly reduced following treatment with the aforementioned three drugs. In contrast to adherent cultures, the spherical cultures were more resistant to the tested drugs, as was reflected by their capacity to re‑create the colonospheres when sustained in serum‑free medium. Flow cytometric analysis of the drug‑treated HCT116 cell‑derived spheres revealed changes in the fraction of cells expressing markers of cancer stem cells (CSCs), whereas the CSC phenotype of HT29 cell‑derived colonospheres was affected to a lesser extent. All reagents enhanced the percentage of non‑viable cells in the colonospheres despite the diminished fraction of active caspase‑3‑positive cells following treatment of the HT29 cell‑derived spheres with anti‑EGFR antibody. Increased autophagy, assessed by acridine orange staining, was noted following the incubation of the HT29‑colonospheres with ASA and 5‑FU in comparison to the control. Notably, the percentage of cyclooxygenase (COX)‑2‑positive cells was not affected by ASA, although its activity was markedly elevated in the colonospheres incubated with anti‑EGFR antibody. On the whole, the findings of this study indicate that all the tested drugs were involved in different cellular processes, which suggests that they should be considered for the combined therapeutic treatment of CRC, particularly for targeting the population of CSC‑like cells. Thus, cancer cell‑derived spheres may be used as a preferable model for in vitro anticancer drug testing.

Synthetic cathinones are psychoactive substances, derivatives of a natural psychostimulant cathinone. Although many synthetic cathinones have lost their legal status in many countries, their abuse still continues worldwide. Recently, they have been reported to exert neurotoxic effects in vitro and in vivo. The molecular mechanisms of their action have not been fully elucidated. Recently, they have been linked to the induction of oxidative stress, autophagy, and apoptosis. The aim of this study was to investigate whether 3-fluoromethcathinone (3-FMC), a synthetic cathinone, is able to induce oxidative stress, autophagy, and apoptosis in HT22 immortalized mouse hippocampal cells. We found that treatment of HT22 cells with this compound results in a concentration-dependent increase in the intracellular production of reactive oxygen species. Moreover, 3-FMC induced concentration-dependent conversion of cytosolic LC3-I to membrane-bound LC3-II and formation of autophagic vacuoles. Additionally, the level of p62/SQSTM1 protein decreased after 3-FMC treatment, suggesting that accumulation of autophagic vacuoles resulted from activation rather than inhibition of autophagy. Our results also showed that 3-FMC at millimolar concentration is able to induce caspase-dependent apoptotic cell death in HT22 cells. Our findings suggest that abuse of 3-FMC may disturb neuronal homeostasis and impair functioning of the central nervous system.

We have proposed a new method which can be applied in assessing the intracellular production of hydrogen peroxide. Using this assay we have examined the hydrogen peroxide generation during the L-glutamate induced oxidative stress in the HT22 hippocampal cells. The detection of hydrogen peroxide is based on two crucial reagents cis-[Cr(C 2 O 4 )(pm)(OH 2 ) 2 ] + (pm denotes pyridoxamine) and 2-ketobutyrate. The results obtained indicate that the presented method can be a promising tool to detect hydrogen peroxide in biological samples, particularly in cellular experimental models.

The purpose of this study was to examine the application of the coordinated cis-[Cr(C2O4)(pm)(OH)2]+ cation where pm denotes pyridoxamine, as a specific sensing ion for the detection of hydrogen peroxide (H2O2). The proposed method for H2O2 detection includes two key steps. The first step is based on the nonenzymatic decarboxylation of pyruvate upon reaction with H2O2, while the second step is based on the interaction of cis-[Cr(C2O4)(pm)(OH2)2]+ with the CO2 released in the previous step. Using this method H2O2 generated during glutamate-induced oxidative stress was detected in HT22 hippocampal cells. The coordination ion cis-[Cr(C2O4)(pm)(OH2)2]+ and the spectrophotometric stopped-flow technique were applied to determine the CO2 concentration in cell lysates, supernatants and cell-free culture medium. Prior to CO2 assessment pyruvate was added to all samples studied. Pyruvate reacts with H2O2 with 1:1 stoichiometry, and consequently the amount of CO2 released in this reaction is equivalent to the amount of H2O2.

Recently, the abuse of recreational drugs has become an important problem in many countries. Among these psychoactive substances are synthetic cathinones, a group of compounds derived from the alkaloid cathinone, that have gained widespread popularity. Many cathinones have demonstrated neurotoxic effects. The aim of this study was to examine the effects of 3-fluoromethcathinone, a structural analog of mephedrone, on HT22 mouse hippocampal cells. Cell viability was assessed using the sulforhodamine B assay. Flow cytometry was used to study the cell cycle distribution. We found that 3-fluoromethcathinone inhibits growth of HT22 cells. Our results also revealed that it induces G0/G1-phase cell cycle arrest. To our knowledge, this is the first study to demonstrate the cytotoxic action of 3-fluoromethcathinone. Our findings suggest that abuse of this cathinone derivative may not be without risk.

Many biologically active compounds, including macromolecules that are used as various kinds of drugs, must be delivered to the interior of cell or organelles such as mitochondria or nuclei to achieve a therapeutic effect. However, very often, lipophilic cell membrane is impermeable for these molecules. A new method in the transport of macromolecules through the cell membrane is the one based on utilizing cell-penetrating peptides (CPPs). Invented 25 years ago, CPPs are currently the subject of intensive research in many laboratories all over the world. CPPs are short compounds comprising up to 30 amino acid residues, which penetrate the cell membrane but do not cause cell damage. Additionally, CPPs can transfer hydrophilic molecules (peptides, proteins, nucleic acids) which exceed their mass, and for which the cell membrane is generally impermeable. In this review, we concentrate on the cellular uptake mechanism of CPPs and a method of conjunction of CPPs to the transported molecules. We also highlight the potential of CPPs in delivering various kinds of macromolecules into cells, including compounds of therapeutic interest.

Introduction: Cell penetrating peptides (CPPs) have the ability to translocate through cell membranes with high efficiency and therefore can introduce biological agents with pharmaceutical properties into the cell. Transportan (TP) and its shorter analog transportan 10 (TP10) are among the best studied CPPs, however, their effects on viability of and cargo introduction into colorectal cancer (CRC) cells have yet not been investigated. The aim of our study was to evaluate the cytotoxic effects of TP and TP10 on representative CRC lines and the efficiency of protein (streptavidin) and siRNA cargo delivery by TP-biotinylated derivatives (TP-biot)., Material and Methods: HT29 (early stage CRC model) and HCT116 (metastatic CRC model) cell lines were incubated with TP, TP10, TP-biot1, TP-biot13 and TP10-biot1. The effects of studied CPPs on cell viability and cell cycle were assessed by MTT and annexin V assays. The uptake of streptavidin-FITC complex into cells was determined by flow cytometry and fluorescence microscopy, with the inhibition of cellular vesicle trafficking by brefeldin A. The efficiency of siRNA for SASH1 gene delivery was measured by quantitative PCR (qPCR)., Results: Since up to 10 µM concentrations of each CPP showed no significant cytotoxic effect, the concentrations of 0.5-5 µM were used for further analyses. Within this concentration range none of the studied CPPs affected cell viability and cell cycle. The efficient and endocytosis-independent introduction of streptavidin-FITC complex into cells was observed for TP10-biot1 and TP-biot1 with the cytoplasmic location of the fluorescent cargo; decreased SASH1 mRNA level was noticed with the use of siRNA and analyzed CPPs., Conclusions: We conclude that TP, TP10 and their biotinylated derivatives can be used as efficient delivery vehicles of small and large cargoes into CRC cells.

Pterostilbene, a naturally occurring structural analog of resveratrol, has been reported to exert antiproliferative and proapoptotic effects in various cancer types. Recently, it has been demonstrated to induce both autophagy and apoptosis in human bladder and breast cancer cell lines. The aim of this study was to evaluate the effects of pterostilbene on HL60 human leukemia cells. Cell morphology was examined using confocal and electron microscopy. Cell viability was determined by MTT, neutral red uptake and trypan blue exclusion assays. LC3 processing was studied based on Western blotting and immunofluorescence analyses. Flow cytometry was used to study cell cycle distribution, phosphatidylserine externalization, caspase activation, disruption of mitochondrial membrane potential and intracellular production of reactive oxygen species. DNA degradation was examined by gel electrophoresis. We found that treatment of HL60 cells with pterostilbene at the IC90 concentration resulted in the G0/G1 cell cycle arrest. Pterostilbene induced conversion of cytosolic LC3-I to membrane-bound LC3-II and accumulation of large LC3-positive vacuolar structures. Pterostilbene also led to phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase activation and disruption of mitochondrial membrane potential. Moreover, it did not induce oxidative stress. Our results suggest that pterostilbene induces accumulation of autophagic vacuoles followed by cell death in HL60 cells.

Merkel cell carcinoma (MCC) is a rare and highly aggressive cutaneous carcinoma with characteristics of neuroendocrine tumor. We performed immunohistochemical analysis to demonstrate the presence of various neuropeptides within cells of MCC resected from a 75-year old woman. The cells of primary tumor of cheek were compared with the cells of regional right submandibular metastatic tumor which was found eight months later. A double- staining IHC for the pan-neuronal marker, PGP 9.5, and selected neuropeptides in the tissue material obtained from both locations was performed. Single multipolar cells in the main mass of primary tumor stained positively for PGP 9.5 and such neuropeptides as GAL, VIP, PACAP, NPY and CGRP. Moreover, we demonstrated for the first time the presence of neuropeptides in metastatic MCC cells. In the metastatic tumor, cells showing the co-localization of PGP-9.5 and neuropeptides were more numerous, mostly of oval shape, and significantly smaller than in the primary tumor. Thus, the progression of MCC may be associated with the acquisition by its cells of new morphological and biological features.

Pterostilbene, a polyphenolic compound present in grapes and other fruits, has been demonstrated to inhibit growth and induce apoptosis and autophagy in some cancer cell types. We found that pterostilbene at the IC(90) concentration of 44 µM inhibited proliferation and induced apoptosis in MOLT4 human leukemia cells. Treatment with pterostilbene resulted in a transient accumulation of cells in the G(0)/G(1)-cell cycle phase followed by the S-phase arrest. Pterostilbene-induced apoptotic death of MOLT4 cells was mediated by caspase-3 activation and was accompanied by the disruption of mitochondrial membrane potential, phosphatidylserine externalization and internucleosomal DNA fragmentation. Our results suggest that pterostilbene could serve as a potential additional chemotherapeutic agent for the treatment of leukemia.