Evaluation of the Biological Effect of Non-UV-Activated Bergapten on Selected Human Tumor Cells and the Insight into the Molecular Mechanism of Its Action.

There is some evidence that non-photoactivated psoralens may be active against breast and colon tumor cells. Therefore, we evaluated the antiproliferative, proapoptotic, and anti-migrative effect of 5-methoxypsoralen (5-MOP) isolated from Peucedanum tauricum MB fruits in human colorectal adenocarcinoma (HT-29 and SW620), osteosarcoma (Saos-2 and HOS), and multiple myeloma (RPMI8226 and U266). Dose- and cell-line-dependent effects of 5-MOP on viability and proliferation were observed, with the strongest inhibitory effect against Saos-2 and a moderate effect against the HOS, HT-29, and SW620 cells. Multiple myeloma showed low sensitivity. The high viability of human normal cell cultures (HSF and hFOB) in a wide range of 5-MOP concentrations tested (6.25-100 µM) was confirmed. Moreover, the migration of treated Saos-2, SW620, and HT-29 cell lines was impaired, as indicated via a wound healing assay. Flow cytometry analysis conducted on Saos-2 cells revealed the ability of 5-MOP to block the cell cycle in the G2 phase and trigger apoptosis, which was accompanied by a loss of mitochondrial membrane potential, caspases (-9 and -3) activation, the altered expression of the Bax and Bcl-2 proteins, and decreased AKT phosphorylation. This is the first report evaluating the antiproliferative and antimigratory impact of non-UV-activated bergapten on the abovementioned (except for HT-29) tumor cells, which provides new data on the potential role of 5-MOP in inhibiting the growth of various types of therapeutic-resistant cancers.

Thalidomide derivatives as nanomolar human neutrophil elastase inhibitors: Rational design, synthesis, antiproliferative activity and mechanism of action.

Here, we rationally designed a human neutrophil elastase (HNE) inhibitors 4a-4f derived from thalidomide. The HNE inhibition assay showed that synthesized compounds 4a, 4b, 4e and 4f demonstrated strong HNE inhibiton properties with IC50 values of 21.78-42.30 nM. Compounds 4a, 4c, 4d and 4f showed a competitive mode of action. The most potent compound 4f shows almost the same HNE inhibition as sivelestat. The molecular docking analysis revealed that the strongest interactions occur between the azetidine-2,4-dione group and the following three aminoacids: Ser195, Arg217 and His57. A high correlation between the binding energies and the experimentally determined IC50 values was also demonstrated. The study of antiproliferative activity against human T47D (breast carcinoma), RPMI 8226 (multiple myeloma), and A549 (non-small-cell lung carcinoma) revealed that designed compounds were more active compared to thalidomide, pomalidomide and lenalidomide used as the standard drugs. Additionally, the most active compound 4f derived from lenalidomide induces cell cycle arrest at the G2/M phase and apoptosis in T47D cells.

Pseudomonas aeruginosa exotoxin A induces apoptosis in Galleria mellonella hemocytes.

The cellular immune response of the greater wax moth Galleria mellonella to Pseudomonas aeruginosa exotoxin A was investigated for the first time. The insects were challenged with a sublethal dose of exoA, and then hemocyte parameters were assessed. The analysis showed a statistically significant decrease in the total hemocyte count (THC), which was associated with significant decreases in the number of granulocytes and plasmatocytes. In turn, no statistically significant changes were observed in the number of spherulocytes and oenocytoides. Fluorescent staining indicated that cells collected from the exoA-challenged larvae exhibited features characteristic for apoptotic and autophagic cell death, e.g. cytoplasm vacuolization and chromatin condensation. The flow cytometry analysis revealed a significant increase in the number of phosphatidylserine- and active caspase 3-positive hemocytes challenged with exoA, which proved apoptosis induction. Our results will help in understanding the role of exotoxin A during P. aeruginosa infections not only in insects but also in mammals, including humans.

Discovery of New 3,3-Diethylazetidine-2,4-dione Based Thiazoles as Nanomolar Human Neutrophil Elastase Inhibitors with Broad-Spectrum Antiproliferative Activity.

A series of 3,3-diethylazetidine-2,4-dione based thiazoles 3a-3j were designed and synthesized as new human neutrophil elastase (HNE) inhibitors in nanomolar range. The representative compounds 3c, 3e, and 3h exhibit high HNE inhibitory activity with IC50 values of 35.02-44.59 nM, with mixed mechanism of action. Additionally, the most active compounds 3c and 3e demonstrate high stability under physiological conditions. The molecular docking study showed good correlation of the binding energies with the IC50 values, suggesting that the inhibition properties are largely dependent on the stage of ligand alignment in the binding cavity. The inhibition properties are correlated with the energy level of substrates of the reaction of ligand with Ser195. Moreover, most compounds showed high and broad-spectrum antiproliferative activity against human leukemia (MV4-11), human lung carcinoma (A549), human breast adenocarcinoma (MDA-MB-231), and urinary bladder carcinoma (UMUC-3), with IC50 values of 4.59-9.86 µM. Additionally, compounds 3c and 3e can induce cell cycle arrest at the G2/M phase and apoptosis via caspase-3 activation, leading to inhibition of A549 cell proliferation. These findings suggest that these new types of drugs could be used to treat cancer and other diseases in which immunoreactive HNE is produced.

New Borane-Protected Derivatives of α-Aminophosphonous Acid as Anti-Osteosarcoma Agents: ADME Analysis and Molecular Modeling, In Vitro Studies on Anti-Cancer Activities, and NEP Inhibition as a Possible Mechanism of Anti-Proliferative Activity.

Many organophosphorus compounds (OPs), especially various α-aminophosphonates, exhibit anti-cancer activities. They act, among others, as inhibitors of the proteases implicated in cancerogenesis. Thesetypes of inhibitors weredescribed, e.g., for neutral endopeptidase (NEP) expressed in different cancer cells, including osteosarcoma (OS). The aim of the present study isto evaluate new borane-protected derivatives of phosphonous acid (compounds 1-7) in terms of their drug-likeness properties, anti-osteosarcoma activities in vitro (against HOS and Saos-2 cells), and use as potential NEP inhibitors. The results revealed that all tested compounds exhibited the physicochemical and ADME properties typical for small-molecule drugs. However, compound 4 did not show capability of blood-brain barrier penetration (Lipinski and Veber rules;SwissAdme tool). Moreover, the α-aminophosphonite-boranes (compounds 4-7) exhibited stronger anti-proliferative activity against OS cells than the other phosphonous acid-borane derivatives (compounds 1-3),especially regarding HOS cells (MTT assay). The most promising compounds 4 and 6 induced apoptosis through the activation of caspase 3 and/or cell cycle arrest at the G2 phase (flow cytometry). Compound 4 inhibited the migration and invasiveness of highly aggressive HOS cells (wound/transwell and BME-coated transwell assays, respectively). Additionally, compound 4 and, to a lesser extent, compound 6 inhibited NEP activity (fluorometric assay). This activity of compound 4 was involved in its anti-proliferative potential (BrdU assay). The present study shows that compound 4 can be considered a potential anti-osteosarcoma agent and a scaffold for the development of new NEP inhibitors.

Alpha Ketoglutarate Downregulates the Neutral Endopeptidase and Enhances the Growth Inhibitory Activity of Thiorphan in Highly Aggressive Osteosarcoma Cells.

Since natural substances are widely explored as epigenetic modulators of gene expression and epigenetic abnormalities are important causes of cancerogenesis, factors with pro-tumor activities subjected to epigenetic control, e.g., neutral endopeptidase (NEP, neprilysin), are promising anticancer targets for potential therapies acting via epigenetic regulation of gene expression. Alpha-ketoglutarate (AKG) is a naturally occurring co-substrate for enzymes involved in histone and DNA demethylation with suggested anti-cancer activity. Hence, we investigated a potential effect of AKG on the NEP expression in cells derived from various cancers (cervical, colon, osteosarcoma) and normal epithelial cells and osteoblasts. Moreover, the overall methylation status of histone H3 was explored to establish the molecular target of AKG activity. Additionally, it was investigated whether AKG in combination with thiorphan (NEP specific inhibitor) exhibited enhanced anticancer activity. The results revealed that AKG downregulated the expression of NEP at the protein level only in highly aggressive osteosarcoma HOS cells (flow cytometry and fluorometric assays), and this protease was found to be involved in AKG-induced growth inhibition in osteosarcoma cells (siRNA NEP silencing, BrdU assay, flow cytometry). Unexpectedly, AKG-induced hypermethylation of H3K27 in HOS cells, which was partially dependent on EZH2 activity. However, this effect was not implicated in the AKG-induced NEP downregulation (flow cytometry). Finally, the combined treatment with AKG and thiorphan was shown to significantly enhance the growth inhibitory potential of each one towards HOS cells (BrdU assay). These preliminary studies have shown for the first time that the downregulation of NEP expression is a promising target in therapies of NEP-implicating HOS cells. Moreover, this therapeutic goal can be achieved via AKG-induced downregulation of NEP and synergistic activity of AKG with thiorphan, i.e., a NEP specific inhibitor. Furthermore, this study has reported for the first time that exogenous AKG can influence the activity of histone methyltransferase, EZH2. However, this issue needs further investigation to elucidate the mechanisms of this phenomenon.

Xanthohumol Impairs the PMA-Driven Invasive Behaviour of Lung Cancer Cell Line A549 and Exerts Anti-EMT Action.

Xanthohumol (XN), the main prenylated flavonoid from hop cones, has been recently reported to exert significant proapoptotic, anti-proliferative, and growth inhibitory effects against lung cancer in both in vitro and in vivo studies. However, its anti-metastatic potential towards this malignancy is still unrevealed. Previously, we indicated that the human lung adenocarcinoma A549 cell line was sensitive to XN treatment. Therefore, using the same tumour cell model, we have studied the influence of XN on the phorbol-12-myristate-13-acetate (PMA)-induced cell migration and invasion. The effects of XN on the expression/activity of pro-invasive MMP-9 and MMP-2 and the expression of MMP inhibitors, i.e., TIMP-1 and TIMP-2 (anti-angiogenic factors), were evaluated. Additionally, the influence of XN on the production of the key pro-angiogenic cytokine, i.e., VEGF, and the release of TGF-ß, which is both a pro-angiogenic cytokine and an epithelial-mesenchymal transition (EMT) stimulator, was studied. Furthermore, the influence of XN on the expression of EMT-associated proteins such as E-cadherin and α-E-catenin (epithelial markers), vimentin and N-cadherin (mesenchymal markers), and Snail-1 (transcriptional repressor of E-cadherin) was studied. To elucidate the molecular mechanism underpinning the XN-mediated inhibition of metastatic progression in PMA-activated cells, the phosphorylation levels of AKT, FAK, and ERK1/2 kinases, which are signalling molecules involved in EMT program activation, were assayed. The results showed that XN in non-cytotoxic concentrations impaired the PMA-driven migratory and invasive capacity of A549 cells by decreasing the level of expression of MMP-9 and concomitantly increasing the expression of the TIMP-1 protein, i.e., a specific blocker of pro-MMP-9 activation. Moreover, XN decreased the PMA-induced production of VEGF and TGF-ß. Furthermore, the XN-treatment counteracted the PMA-induced EMT of the A549 cells by the upregulation of E-cadherin and α-E-catenin and the downregulation of N-cadherin, vimentin, and Snail-1 expression. The proposed mechanism underlying the anti-invasive XN activity involved the inhibition of the ERK/MAPK pathway and suppression of FAK and PI3/AKT signalling. Our results suggesting migrastatic properties of XN against lung cancer cells require further verification in in vivo assays.

Alpha Ketoglutarate Exerts In Vitro Anti-Osteosarcoma Effects through Inhibition of Cell Proliferation, Induction of Apoptosis via the JNK and Caspase 9-Dependent Mechanism, and Suppression of TGF-ß and VEGF Production and Metastatic Potential of Cells.

Osteosarcoma (OS) is the most common type of primary bone tumor. Currently, there are limited treatment options for metastatic OS. Alpha-ketoglutarate (AKG), i.e., a multifunctional intermediate of the Krebs cycle, is one of the central metabolic regulators of tumor fate and plays an important role in cancerogenesis and tumor progression. There is growing evidence suggesting that AKG may represent a novel adjuvant therapeutic opportunity in anti-cancer therapy. The present study was intended to check whether supplementation of Saos-2 and HOS osteosarcoma cell lines (harboring a TP53 mutation) with exogenous AKG exerted an anti-cancer effect. The results revealed that AKG inhibited the proliferation of both OS cell lines in a concentration-dependent manner. As evidenced by flow cytometry, AKG blocked cell cycle progression at the G1 stage in both cell lines, which was accompanied by a decreased level of cyclin D1 in HOS and increased expression of p21Waf1/Cip1 protein in Saos-2 cells (evaluated with the ELISA method). Moreover, AKG induced apoptotic cell death and caspase-3 activation in both OS cell lines (determined by cytometric analysis). Both the immunoblotting and cytometric analysis revealed that the AKG-induced apoptosis proceeded predominantly through activation of an intrinsic caspase 9-dependent apoptotic pathway and an increased Bax/Bcl-2 ratio. The apoptotic process in the AKG-treated cells was mediated via c-Jun N-terminal protein kinase (JNK) activation, as the specific inhibitor of this kinase partially rescued the cells from apoptotic death. In addition, the AKG treatment led to reduced activation of extracellular signal-regulated kinase (ERK1/2) and significant inhibition of cell migration and invasion in vitro concomitantly with decreased production of pro-metastatic transforming growth factor ß (TGF-ß) and pro-angiogenic vascular endothelial growth factor (VEGF) in both OS cell lines suggesting the anti-metastatic potential of this compound. In conclusion, we showed the anti-osteosarcoma potential of AKG and provided a rationale for a further study of the possible application of AKG in OS therapy.

Tropinone-Derived Alkaloids as Potent Anticancer Agents: Synthesis, Tyrosinase Inhibition, Mechanism of Action, DFT Calculation, and Molecular Docking Studies.

A new series of hybrid compounds with tropinone and thiazole rings in the structure was designed and synthesized as potential anticancer agents. They were tested against human multiple myeloma (RPMI 8226), lung carcinoma (A549), breast adenocarcinoma (MDA-MB-231), and mouse skin melanoma (B16-F10) cell lines. Toxicity was tested on human normal skin fibroblasts (HSF) and normal colon fibroblasts (CCD-18Co). The growth inhibition mechanism of the most active derivative was analyzed through investigation of its effect on the distribution of cell cycle phases and ability to induce apoptosis and necrosis in RPMI 8226 and A549 cancer cells. The tyrosinase inhibitory potential was assessed, followed by molecular docking studies. Compounds 3a-3h show high anticancer activity against MDA-MB-231 and B16-F10 cell lines with IC50 values of 1.51-3.03 µM. Moreover, the cytotoxic activity of the investigated compounds against HSF and CCD-18Co cells was 8-70 times lower than against the cancer cells or no toxicity was shown in our tests, with derivative 3a being particularly successful. The mechanism of action of compound 3a in RPMI 8226 cell was shown to be through induction of cell death through apoptosis. The derivatives show ability to inhibit the tyrosinase activity with a mixed mechanism of inhibition. The final molecular docking results showed for IC50 distinct correlation with experiment.

Xanthohumol exhibits anti-myeloma activity in vitro through inhibition of cell proliferation, induction of apoptosis via the ERK and JNK-dependent mechanism, and suppression of sIL-6R and VEGF production.

BACKGROUND: Xanthohumol (XN, a hop-derived prenylflavonoid) was found to exert anticancer effects on various cancer types. However, the mechanisms by which XN affects the survival of multiple myeloma cells (MM) are little known. Therefore, our study was undertaken to address this issue. METHODS: Anti-proliferative activity of XN towards two phenotypically distinct MM cell lines U266 and RPMI8226 was evaluated with the MTT and BrdU assays. Cytotoxicity was determined with the LDH method, whereas apoptosis was assessed by flow cytometry and fluorescence staining. The expression of cell cycle- and apoptosis-related proteins and the activation status of signaling pathways were estimated by immunoblotting and ELISA assays. RESULTS: XN reduced the viability of RPMI8226 cells more potently than in U266 cells. It blocked cell cycle progression through downregulation of cyclin D1 and increased p21 expression. The marked apoptosis induction in the XN-treated RPMI8226 cells was related to initiation of mitochondrial and extrinsic pathways, as indicated by the altered p53, Bax, and Bcl-2 protein expression, cleavage of procaspase 8 and 9, and elevated caspase-3 activity. The apoptotic process was probably mediated via ROS overproduction and MAPK (ERK and JNK) activation as N-acetylcysteine, or specific inhibitors of these kinases prevented the XN-induced caspase-3 activity and, hence, apoptosis. Moreover, XN decreased sIL-6R and VEGF production in the studied cells. CONCLUSIONS: ERK and JNK signaling pathways are involved in XN-induced cytotoxicity against MM cells. GENERAL SIGNIFICANCE: The advanced understanding of the molecular mechanisms of XN action can be useful in developing therapeutic strategies to treat multiple myeloma.

Betulin Promotes Differentiation of Human Osteoblasts In Vitro and Exerts an Osteoinductive Effect on the hFOB 1.19 Cell Line Through Activation of JNK, ERK1/2, and mTOR Kinases.

Although betulin (BET), a naturally occurring pentacyclic triterpene, has a variety of biological activities, its osteogenic potential has not been investigated so far. The aim of this study was to assess the effect of BET on differentiation of human osteoblasts (hFOB 1.19 and Saos-2 cells) in vitro in osteogenic (with ascorbic acid as an osteogenic supplement) and osteoinductive (without an additional osteogenic supplement) conditions. Osteoblast differentiation was evaluated based on the mRNA expression (RT-qPCR) of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), type I collagen-α1 (COL1A1), and osteopontin (OPN). Additionally, ALP activity and production of COL1A1 (western blot analysis) and OPN (ELISA) were evaluated. The level of mineralization (calcium accumulation) was determined with Alizarin red S staining. BET upregulated the mRNA level of RUNX2 and the expression of other osteoblast differentiation markers in both cell lines (except the influence of BET on ALP expression/activity in the Saos-2 cells). Moreover, it increased mineralization in both cell lines in the osteogenic conditions. BET also increased the mRNA level of osteoblast differentiation markers in both cell lines (except for ALP in the Saos-2 cells) in the osteoinductive conditions, which was accompanied with increased matrix mineralization. The osteoinductive activity of BET in the hFOB 1.19 cells was probably mediated via activation of MAPKs (JNK and ERK1/2) and mTOR, as the specific inhibitors of these kinases abolished the BET-induced osteoblast differentiation. Our results suggest that BET has the potential to enhance osteogenesis.

Alpha ketoglutarate exerts a pro-osteogenic effect in osteoblast cell lines through activation of JNK and mTOR/S6K1/S6 signaling pathways.

Although numerous in vivo studies have suggested that alpha-ketoglutarate (AKG), i.e. the key intermediate in the Krebs cycle, may have an anabolic effect on bone tissue, the direct influence of AKG on osteoblasts and the underlying mechanism of its action have not been investigated so far. The aim of this study was to assess the impact of AKG (disodium salt dihydrate) on osteogenesis in vitro and identification of some signaling mechanisms involved in this activity. The human and mouse normal osteoblast cell lines hFOB 1.19 and MC3T3-E1 were used in this study. The results showed that AKG did not increase the proliferation of osteoblasts; however, it upregulated the expression of transcription factors RUNX2 and Osterix, the mRNA and protein levels of osteoblast differentiation markers (alkaline phosphatase, type I collagen, bone sialoprotein II, osteopontin, osteocalcin), and the mineralization levels in the hFOB 1.19 and MC3T3-E1 cell cultures. Moreover, AKG increased JNK, mTOR, S6K1, and S6 phosphorylation and decreased ERK1/2 phosphorylation in both osteoblast cell lines. The JNK inhibitor and rapamycin, but not the ERK inhibitor, abolished the AKG-promoted osteoblast differentiation. Using immunofluorescence staining, qRT-PCR, and Western blot analysis, we detected the presence of an AKG receptor GPR99 activated by alpha ketoglutaric acid in the tested osteoblast cell lines. However, AKG salt did not activate GPR99. Our findings suggest that AKG salt activates the JNK and mTOR/S6K1/S6 signaling pathways to promote differentiation of osteoblasts, independently of GPR99 activation. We can conclude that AKG salts might be promising candidates for bone anabolic drugs used for prevention or/and treatment of osteoporosis.

Neutral endopeptidase (NEP) inhibitors - thiorphan, sialorphin, and its derivatives exert anti-proliferative activity towards colorectal cancer cells in vitro.

Neutral endopeptidase (NEP) is an enzyme implicated in development of different tumors, e.g. colorectal cancer (CRC). In this study, the anti-cancer effects of NEP inhibitors, thiorphan (synthetic compound) and sialorphin (naturally occurring pentapeptide) on CRC cells were investigated. Moreover, we synthesized some derivatives of sialorphin (alanine scan analogues: AHNPR, QANPR, QHAPR, QHNAR; N-acetylated sialorphin; C-amidated sialorphin, and C-amidated alanine scan analogues) to examine the biological activity of these inhibitors on CRC cells. The cytotoxic activity of the NEP inhibitors against CRC cell lines (SW620 and LS180) and normal human fibroblasts (HSF) was evaluated. Additionally, the influence of NEP inhibitors on proliferation, cell cycle progression, induction of apoptosis, and the level of phosphorylation of MAP kinases and mTORC1 signaling pathway proteins in CRC cells were examined. The NEP inhibitors were non-cytotoxic to HSF cells; however, most of them slightly decreased the viability and inhibited proliferation of CRC cells. The N-acetylation or C-amidation of sialorphin or its alanine scan analogues resulted in decreased or abolished anti-proliferative activity of the NEP inhibitors towards the CRC cells. Additionally, thiorphan and sialorphin enhanced the anti-proliferative activity of other CRC-cell growth inhibitors (atrial natriuretic peptide-ANP and melphalan-MEL). The mechanisms involved in the anti-proliferative effects of the tested inhibitors were mediated via NEP and associated with induction of cell cycle arrest in the G0/G1 phase, increased activity of ERK1/2, and a reduced level of phosphorylation of mTOR (Ser2448), 4E-BP1, and p70S6K. However, the NEP inhibitors did not induce apoptosis in the CRC cells. These results have indicated that thiorphan and sialorphin or its derivatives AHNPR, QANPR, QHAPR, and QHNAR have the potential to be used as agents in treatment of patients with CRC.

Neutral endopeptidase (NEP) is differentially involved in biological activities and cell signaling of colon cancer cell lines derived from various stages of tumor development.

The presented studies were aimed at exploring the role of neutral endopeptidase (NEP) in the function of colon cancer cell lines LS 180 and SW 620, derived from different grades and stages of tumor development. NEP silencing by siRNA resulted in decreased viability and proliferation accompanied by increased apoptosis in both cell lines. Additionally, cell cycle arrest at the G2/M phase was observed, but only in LS 180 cells. Opposite to these results, serum-stimulated migration was increased in both cell lines. Furthermore, NEP silencing influenced the invasive activity of LS 180 and SW 620 cells in an opposite manner: while LS 180 cells showed an enhanced invasiveness, SW 620 cells exerted a reduced activity. An exploration of the activity of signaling molecules responsible for the function of tumor cells-Akt, PTEN, and FAK-after NEP silencing indicated that the endopeptidase is involved in their regulation. The increased phosphorylation level of Akt was accompanied by a decrease in PTEN in the presence of a high concentration of serum. A reduced concentration of serum did not change the phosphorylation status of Akt. Enhanced autophosphorylation of FAK was observed in LS 180 and SW 620 cells cultivated in a medium with a high concentration of serum. Taken together, these results confirm that NEP is implicated in the regulation of the survival, growth, and motile activity of colon cancer. This is also the first report which shows that NEP mediates cancer cell migration and invasiveness, but not growth and survival, through Akt/FAK signaling pathways.