Quinoline-based thiazolyl-hydrazones target cancer cells through autophagy inhibition.

Heterocyclic pharmacophores such as thiazole and quinoline rings have a significant role in medicinal chemistry. They are considered privileged structures since they constitute several Food and Drug Administration (FDA)-approved drugs for cancer treatment. Herein, we report the synthesis, in silico evaluation of the ADMET profiles, and in vitro investigation of the anticancer activity of a series of novel thiazolyl-hydrazones based on the 8-quinoline (1a-c), 2-quinoline (2a-c), and 8-hydroxy-2-quinolyl moiety (3a-c). The panel of several human cancer cell lines and the nontumorigenic human embryonic kidney cell line HEK-293 were used to evaluate the compound-mediated in vitro anticancer activities, leading to [2-(2-(quinolyl-8-ol-2-ylmethylene)hydrazinyl)]-4-(4-methoxyphenyl)-1,3-thiazole (3c) as the most promising compound. The study revealed that 3c blocks the cell-cycle progression of a human colon cancer cell line (HCT-116) in the S phase and induces DNA double-strand breaks. Also, our findings demonstrate that 3c accumulates in lysosomes, ultimately leading to the cell death of the hepatocellular carcinoma cell line (Hep-G2) and HCT-116 cells, by the mechanism of autophagy inhibition.

Correlating Basal Gene Expression across Chemical Sensitivity Data to Screen for Novel Synergistic Interactors of HDAC Inhibitors in Pancreatic Carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies. Development of the chemoresistance in the PDAC is one of the key contributors to the poor survival outcomes and the major reason for urgent development of novel pharmacological approaches in a treatment of PDAC. Systematically tailored combination therapy holds the promise for advancing the treatment of PDAC. However, the number of possible combinations of pharmacological agents is too large to be explored experimentally. In respect to the many epigenetic alterations in PDAC, epigenetic drugs including histone deacetylase inhibitors (HDACi) could be seen as the game changers especially in combined therapy settings. In this work, we explored a possibility of using drug-sensitivity data together with the basal gene expression of pancreatic cell lines to predict combinatorial options available for HDACi. Developed bioinformatics screening protocol for predictions of synergistic drug combinations in PDAC identified the sphingolipid signaling pathway with associated downstream effectors as a promising novel targets for future development of multi-target therapeutics or combined therapy with HDACi. Through the experimental validation, we have characterized novel synergism between HDACi and a Rho-associated protein kinase (ROCK) inhibitor RKI-1447, and between HDACi and a sphingosine 1-phosphate (S1P) receptor agonist fingolimod.

Nischarin expression may have differing roles in male and female melanoma patients.

Due to the development of resistance to previously effective therapies, there is a constant need for novel treatment modalities for metastatic melanoma. Nischarin (NISCH) is a druggable scaffolding protein reported as a tumor suppressor and a positive prognostic marker in breast and ovarian cancers through regulation of cancer cell survival, motility and invasion. The aim of this study was to examine the expression and potential role of nischarin in melanoma. We found that nischarin expression was decreased in melanoma tissues compared to the uninvolved skin, and this was attributed to the presence of microdeletions and hyper-methylation of the NISCH promoter in the tumor tissue. In addition to the previously reported cytoplasmic and membranous localization, we observed nischarin in the nuclei in melanoma patients' tissues. NISCH expression in primary melanoma had favorable prognostic value for female patients, but, unexpectedly, high NISCH expression predicted worse prognosis for males. Gene set enrichment analysis suggested significant sex-related disparities in predicted association of NISCH with several signaling pathways, as well as with different tumor immune infiltrate composition in male and female patients. Taken together, our results imply that nischarin may have a role in melanoma progression, but that fine-tuning of the pathways it regulates is sex-dependent. KEY MESSAGES: Nischarin is a tumor suppressor whose role has not been investigated in melanoma. Nischarin expression was downregulated in melanoma tissue compared to the normal skin. Nischarin had the opposite prognostic value in male and female melanoma patients. Nischarin association with signaling pathways differed in females and males. Our findings challenge the current view of nischarin as a universal tumor suppressor.

Synthesis, characterization, and in vivo evaluation of the anticancer activity of a series of 5- and 6-(halomethyl)-2,2'-bipyridine rhenium tricarbonyl complexes.

We report the synthesis, characterization, and in vivo evaluation of the anticancer activity of a series of 5- and 6-(halomethyl)-2,2'-bipyridine rhenium tricarbonyl complexes. The study was promoted in order to understand if the presence and position of a reactive halomethyl substituent on the diimine ligand system of fac-[Re(CO)3]+ species may be a key molecular feature for the design of active and non-toxic anticancer agents. Only compounds potentially able to undergo ligand-based alkylating reactions show significant antiproliferative activity against colorectal and pancreatic cell lines. Of the new species presented in this study, one compound (5-(chloromethyl)-2,2'-bipyridine derivative) shows significant inhibition of pancreatic tumour growth in vivo in zebrafish-Panc-1 xenografts. The complex is noticeably effective at 8 µM concentration, lower than its in vitro IC50 values, being also capable of inhibiting in vivo cancer cells dissemination.

Discovery of 1-Benzhydryl-Piperazine-Based HDAC Inhibitors with Anti-Breast Cancer Activity: Synthesis, Molecular Modeling, In Vitro and In Vivo Biological Evaluation.

Isoform-selective histone deacetylase (HDAC) inhibition is promoted as a rational strategy to develop safer anti-cancer drugs compared to non-selective HDAC inhibitors. Despite this presumed benefit, considerably more non-selective HDAC inhibitors have undergone clinical trials. In this report, we detail the design and discovery of potent HDAC inhibitors, with 1-benzhydryl piperazine as a surface recognition group, that differ in hydrocarbon linker. In vitro HDAC screening identified two selective HDAC6 inhibitors with nanomolar IC50 values, as well as two non-selective nanomolar HDAC inhibitors. Structure-based molecular modeling was employed to study the influence of linker chemistry of synthesized inhibitors on HDAC6 potency. The breast cancer cell lines (MDA-MB-231 and MCF-7) were used to evaluate compound-mediated in vitro anti-cancer, anti-migratory, and anti-invasive activities. Experiments on the zebrafish MDA-MB-231 xenograft model revealed that a novel non-selective HDAC inhibitor with a seven-carbon-atom linker exhibits potent anti-tumor, anti-metastatic, and anti-angiogenic effects when tested at low micromolar concentrations.

The Effects of Resveratrol-Rich Extracts of Vitis vinifera Pruning Waste on HeLa, MCF-7 and MRC-5 Cells: Apoptosis, Autophagia and Necrosis Interplay.

Resveratrol is a well-studied plant-derived molecule in cancer biology, with a plethora of documented in vitro effects. However, its low bioavailability and toxicity risk hamper its wider use. In this study, vine shoots after pruning were used as a source of resveratrol (RSV). The activity of subcritical water extract (SWE) and dry extract (DE) is examined on three cell lines: HeLa, MCF-7 and MRC-5. The cytotoxic effect is assessed by the MTT test and EB/AO staining, levels of apoptosis are determined by Annexin V assay, autophagia by ULK-1 expression using Western blot and NF-kB activation by p65 ELISA. Our results show that both resveratrol-rich extracts (DE, SWE) have a preferential cytotoxic effect on malignant cell lines (HeLa, MCF-7), and low cytotoxicity on non-malignant cells in culture (MRC-5). Further experiments indicate that the investigated malignant cells undergo different cell death pathways. MCF-7 cells died preferentially by apoptosis, while the HeLa cells died most likely by necrosis (possibly ferroptosis). Protective autophagia is diminished upon treatment with DE in both HeLa and MCF-7 cells, while SWE does not influence the level of autophagia. The extracts are effective even at low concentrations (below IC50) in the activation of NF-kB (p65 translocation).

Increased circulating TGF-ß1 is associated with impairment in NK cell effector functions in metastatic melanoma patients.

Transforming growth factor beta (TGF-ß) plays a complex role in carcinogenesis. In 30 melanoma patients and 20 healthy controls (HC) we analysed functional and phenotypic characteristics of NK cells by Flow cytometry, gene expression of TGF-ß1 in peripheral blood mononuclear cells by qPCR and serum and supernatant level of free TGF-ß1 by ELISA. Melanoma patients had significantly higher serum level of circulatingTGF-ß1 compared to HC, especially those with metastasis into the central nervous system (subclass M1d) and high LDH serum values. Melanoma patients compared to HC had significantly higher level of TGF-ß1 gene in PBMC. TGF-ß1 serum values negatively correlate with NK cell activity analysed by CD107a (degranulation marker), IFN-γ, NKG2D, and NKp46 in patients. Study shows the association of high level of TGF-ß1 with NK cell inhibition in patients represents the main mechanism of tumour immune evasion. Targeting TGF-ß may become an important cancer treatment for improving antitumor immunity.

Expanding the Accessible Chemical Space of SIRT2 Inhibitors through Exploration of Binding Pocket Dynamics.

Considerations of binding pocket dynamics are one of the crucial aspects of the rational design of binders. Identification of alternative conformational states or cryptic subpockets could lead to the discovery of completely novel groups of the ligands. However, experimental characterization of pocket dynamics, besides being expensive, may not be able to elucidate all of the conformational states relevant for drug discovery projects. In this study, we propose the protocol for computational simulations of sirtuin 2 (SIRT2) binding pocket dynamics and its integration into the structure-based virtual screening (SBVS) pipeline. Initially, unbiased molecular dynamics simulations of SIRT2:inhibitor complexes were performed using optimized force field parameters of SIRT2 inhibitors. Time-lagged independent component analysis (tICA) was used to design pocket-related collective variables (CVs) for enhanced sampling of SIRT2 pocket dynamics. Metadynamics simulations in the tICA eigenvector space revealed alternative conformational states of the SIRT2 binding pocket and the existence of a cryptic subpocket. Newly identified SIRT2 conformational states outperformed experimentally resolved states in retrospective SBVS validation. After performing prospective SBVS, compounds from the under-represented portions of the SIRT2 inhibitor chemical space were selected for in vitro evaluation. Two compounds, NDJ18 and NDJ85, were identified as potent and selective SIRT2 inhibitors, which validated the in silico protocol and opened up the possibility for generalization and broadening of its application. The anticancer effects of the most potent compound NDJ18 were examined on the triple-negative breast cancer cell line. Results indicated that NDJ18 represents a promising structure suitable for further evaluation.

Targeting Histone Deacetylases: Opportunities for Cancer Treatment and Chemoprevention.

The dysregulation of gene expression is a critical event involved in all steps of tumorigenesis. Aberrant histone and non-histone acetylation modifications of gene expression due to the abnormal activation of histone deacetylases (HDAC) have been reported in hematologic and solid types of cancer. In this sense, the cancer-associated epigenetic alterations are promising targets for anticancer therapy and chemoprevention. HDAC inhibitors (HDACi) induce histone hyperacetylation within target proteins, altering cell cycle and proliferation, cell differentiation, and the regulation of cell death programs. Over the last three decades, an increasing number of synthetic and naturally derived compounds, such as dietary-derived products, have been demonstrated to act as HDACi and have provided biological and molecular insights with regard to the role of HDAC in cancer. The first part of this review is focused on the biological roles of the Zinc-dependent HDAC family in malignant diseases. Accordingly, the small-molecules and natural products such as HDACi are described in terms of cancer therapy and chemoprevention. Furthermore, structural considerations are included to improve the HDACi selectivity and combinatory potential with other specific targeting agents in bifunctional inhibitors and proteolysis targeting chimeras. Additionally, clinical trials that combine HDACi with current therapies are discussed, which may open new avenues in terms of the feasibility of HDACi's future clinical applications in precision cancer therapies.

Antibody VH domain sequence analysis by a bioinformatics approach based on electronic amino acid properties may help to predict paratop location.

Gene as the basic functional unit of DNA encodes information about the product such as protein. The majority of proteins realize function through protein-protein interactions involving short protein motifs. However, some proteins such as antibodies are established by the rearrangement of several (V-D-J) gene segments with the potential addition of nontemplated nucleotides that may change information encoded by the respective gene segment used. Antibody VH domain sequence analysis by ISM bioinformatics approach that is based on amino acids physicochemical features, enable to distinguish the contribution of the information encoded by VH gene or generated during VDJ gene recombination for antibody-antigen interaction. The data presented in this report revealed the significance of CDRH3 for the interaction of antibody specific for immunogenic molecules while CDRH3 contribution is minor for antibody interaction with nonimmunogenic molecules such as haptens and native mammalian dsDNA. Thus, paratopes might be located in the CDRH3 or VH regions.

In Vitro Study of Two Edible Polygonoideae Plants: Phenolic Profile, Cytotoxicity, and Modulation of Keap1-Nrf2 Gene Expression.

Polygonum aviculare and Persicaria amphibia (subfam. Polygonoideae) are used in traditional cuisines and folk medicine in various cultures. Previous studies indicated that phytochemicals obtained from Polygonoideae plants could sensitize chemoresistant cancer cells and enhance the efficacy of some cytostatics. Here, the cytotoxic properties of chemically characterized ethanol extracts obtained from P. aviculare and P. amphibia, individually and in combination with doxorubicin (D), were determined against hepatocarcinoma HepG2 cells. Phenolic composition, cell viability, cell cycle, apoptosis, and the expression of Keap1 and Nrf2 were examined by following methods: LC-MS/MS, LC-DAD-MS, MTT, flow cytometry, and qRT-PCR. Extracts were rich in dietary polyphenolics. Synergistic cytotoxicity was detected for extracts combined with D. The observed synergisms are linked to the interference with apoptosis, cell cycle, and expression of Keap1-Nrf2 genes involved in cytoprotection. The combined approach of extracts and D could emerge as a potential pathway of chemotherapy improvement.

Paclitaxel inhibits transforming growth factor-ß-increased urokinase-type plasminogen activator expression through p38 MAPK and RAW 264.7 macrophage migration.

PURPOSE: Transforming growth factor-ß (TGF-ß) induces alternative macrophage activation that favors tumor progression and immunosuppression. Meanwhile, paclitaxel (PTx) induces macrophage (Mφ) polarization towards antitumor phenotype. TGF-ß also increases tumor stroma macrophage recruitment by mechanisms that include cell motility enhancement and extracellular matrix degradation. In this study, we aimed to determine whether PTx regulates macrophage migration and urokinase-type plasminogen activator (uPA) expression induced by TGF-ß. METHODS: We used mouse macrophage RAW 264.7 cells treated with PTx and TGF-ß combinations. Proliferation was analyzed by MTT and cell cycle assays. Immunofluorescence was performed to determine tubulin cytoskeleton and Smad3 nuclear localization. Western blot and transcriptional luciferase reporters were used to measure signal transduction activation. Migration was determined by wound healing assay. uPA activity was determined by zymography assay. RESULTS: PTx decreased RAW 264.7 cell proliferation by inducing G2/M cell cycle arrest and profoundly modified the tubulin cytoskeleton. Also, PTx inhibited TGF-ß-induced Smad3 activation. Furthermore, PTx decreased cell migration and uPA expression stimulated by TGF-ß. Remarkably, p38 MAPK mediated PTx inhibition of uPA activity induced by TGF-ß but it was not implicated on cell migration inhibition. CONCLUSIONS: PTx inhibits TGF-ß induction of mouse Mφ migration and uPA expression, suggesting that PTx, as TGF-ß targeting therapy, may enhance Mφ anticancer action within tumors.

Telmisartan induces melanoma cell apoptosis and synergizes with vemurafenib in vitro by altering cell bioenergetics.

OBJECTIVE: Despite recent advancements in targeted therapy and immunotherapies, prognosis for metastatic melanoma patients remains extremely poor. Development of resistance to previously effective treatments presents a serious challenge and new approaches for melanoma treatment are urgently needed. The objective of this study was to examine the effects of telmisartan, an AGTR1 inhibitor and a partial agonist of PPARγ, on melanoma cells as a potential agent for repurposing in melanoma treatment. METHODS: Expression of AGTR1 and PPARγ mRNA in melanoma patient tumor samples was examined in publicly available datasets and confirmed in melanoma cell lines by qRT-PCR. A panel of melanoma cell lines was tested in viability, apoptosis and metabolic assays in presence of telmisartan by flow cytometry and immunocytochemistry. A cytotoxic effect of combinations of telmisartan and targeted therapy vemurafenib was examined using the Chou-Talalay combination index method. RESULTS: Both AGTR1 and PPARγ mRNA were expressed in melanoma patient tumor samples and decreased compared to the expression in the healthy skin. In vitro, we found that telmisartan decreased melanoma cell viability by inducing cell apoptosis. Increased glucose uptake, but not utilization, in the presence of telmisartan caused the fission of mitochondria and release of reactive oxygen species. Telmisartan altered the cell bioenergetics, thereby synergizing with vemurafenib in vitro, and even sensitized vemurafenib-resistant cells to the treatment. CONCLUSIONS: Given that the effective doses of telmisartan examined in our study can be administered to patients and that telmisartan is a widely used and safe antihypertensive drug, our findings provide the scientific rationale for testing its efficacy in treatment of melanoma progression.

A Model for Potential B-cell Precursors of Broadly Neutralizing HIV-1 Antibodies Selection and Antibody Affinity Maturation

The goal of this report was to propose a model, wherein synergy between the B-cell antigen receptor (BCR) and toll-like receptor (TLR) signaling is involved in the selection of the B-cell precursors of HIV-1 broadly neutralizing antibodies (bnAbs) with long heavy chain complementarity determining regions 3, from immature/transitional B cells. The model predicts the involvement of Ab/HIV-1 complexes in a way that Ab from the complex binds both BCRs and HIV-1, while on internalization of HIV-1 TLR ligands such as CpG motifs interacts with TLR9. The result of BCR and TLR9 orchestrated signaling is a formation of somatically mutated memory B cells potential precursors of bnAbs. Generated memory B cells continuously exposed to different Ab/HIV-1 complexes can elicit specific bnAb by stochastic somatic hypermutation rather than in the Darwinian process. This new view of the interaction between Ab/HIV-1 complexes and immune system, leading to affinity maturation of the bnAbs in the absence of nominal HIV-1 antigen and BCR interaction, may have implication for the vaccine designed and passive immunization.

Antibody Epitope Specificity for dsDNA Phosphate Backbone Is an Intrinsic Property of the Heavy Chain Variable Germline Gene Segment Used.

Analysis of protein sequences by the informational spectrum method (ISM) enables characterization of their specificity according to encoded information represented with defined frequency (F). Our previous data showed that F(0.367) is characteristic for variable heavy chain (VH) domains (a combination of variable (V), diversity (D) and joining (J) gene segments) of the anti-phosphocholine (PC) T15 antibodies and mostly dependent on the CDR2 region, a site for PC phosphate group binding. Because the T15 dsDNA-reactive U4 mutant also encodes F(0.367), we hypothesized that the same frequency may also be characteristic for anti-DNA antibodies. Data obtained from an analysis of 60 spontaneously produced anti-DNA antibody VH domain sequences supported our hypothesis only for antibodies, which use V gene segment in germline configuration, such as S57(VH31), MRL-DNA22, and VH11, members of the VH1 (J558) and VH7 (S107) gene families. The important finding is that out of seven V gene segments used by spontaneous anti-DNA antibodies, F(0.367) is only expressed by the germline configuration of these three V gene segments. The data suggest that antibody specificity for the phosphate group moiety delineated as F(0.367) is the intrinsic property of the V germline gene segments used, whereas paratope/epitope interaction with antigens bearing this epitope, such as PC or dsDNA, requires corresponding antibody VH conformation that is susceptible to somatic mutation(s).

Author Correction: Iscador Qu inhibits doxorubicin-induced senescence of MCF7 cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Benzothiazole carbamates and amides as antiproliferative species.

A series of new benzothiazole-based carbamates and amides were synthesized and their antiproliferative activity was determined. Derivatives with profound activity were identified and further investigated for their possible mechanism of action. It was found that these compounds induce specific apoptosis, G2/M cell cycle arrest and decrease ROS level in MCF-7 human breast cancer cell line. Moreover, submicromolar antiproliferative activity of examined carbamates against NT2/D1 testicular embryonal carcinoma was shown. The most potent derivatives strongly inhibited NT2/D1 cell migration and invasiveness.

Novel O-methyl goniofufurone and 7-epi-goniofufurone derivatives: synthesis, in vitro cytotoxicity and SAR analysis.

Novel goniofufurone (1) and 7-epi-goniofufurone (2) derivatives bearing a methoxy group at the C-5 and/or C-7 positions were prepared and their in vitro antitumour activity against some human tumour cell lines was evaluated. Some of the analogues displayed powerful antiproliferative effects against the studied tumour cells, but almost all of them were non-cytotoxic toward the normal cells (MRC-5). A SAR study reveals that the introduction of a methoxy group at the C-7 position may increase the antiproliferative effects of the analogues. The most active compounds are 7-O-methyl derivatives of goniofufurone (3) and 7-epi-(+)-goniofufurone (6), which exhibited 1177- and 451-fold higher potencies than the leads 1 and 2 toward the MDA-MB 231 cell line. At the same time, compound 3 is almost 1.5-fold more active than the commercial drug doxorubicin (DOX) against the same cell line. Flow cytometry data confirmed that the cytotoxic effects of these analogues are mediated by apoptosis, additionally revealing that these molecules induced changes in the K562 cell cycle distribution.

Chemical characterization, antioxidant, genotoxic and in vitro cytotoxic activity assessment of Juniperus communis var. saxatilis.

Chemical composition and antioxidative, genotoxic and cytotoxic potential of essential oil (EO) and post-distillation waste (PDW) of Serbian Juniperus communis L. var. saxatilis Pall. was studied in human lung carcinoma (A549) and normal lung fibroblast (MRC-5) cells. GC-MS analysis identified 93.95% of total EO content and determined α-pinen as a dominant component (23.61%). LC-MS/MS analysis of PDW pointed at rutin (12.2 mg g-1) and quinic acid (11.1 mg g-1) as the most abundant. Antioxidativity of PDW was strong in DPPH (IC50 was 5.27 µg mL-1), and moderate in TBA and FRAP assays. Both substances were more cytotoxic to A549 than to MRC-5 cells. Obtained IC50 values were 69.4 µg mL-1 and 120 µg mL-1 for EO, and 1.27 mg mL-1 and 2.86 mg mL-1 for PDW, respectively. PDW was genotoxic (0.3 mg mL-1 and 1 mg mL-1 in A549 and MRC-5 cells, respectively) and induced apoptosis and arrested cell cycle in G2/M phase in A549 cells (0.3 mg mL-1). In mixtures with doxorubicin cytotoxicity of EO and PDW increased, and combination index values (0.12-0.18) revealed clear synergistic effect, stronger in cancer cells. This indicates that J. communis var. saxatilis could decrease the chemotherapeutic doses of doxorubicin, potentially reducing its side effects.

Targeted therapy and personalized medicine in hepatocellular carcinoma: drug resistance, mechanisms, and treatment strategies.

Hepatocellular carcinoma (HCC) is characterized by a growing number of new cases diagnosed each year that is nearly equal to the number of deaths from this cancer. In a majority of the cases, HCC is associated with the underlying chronic liver disease, and it is diagnosed in advanced stage of disease when curative treatment options are not applicable. Sorafenib is a treatment of choice for patients with performance status 1 or 2 and/or macrovascular invasion or extrahepatic spread, and regorafenib is the only systemic treatment found to provide survival benefit in HCC patients progressing on sorafenib treatment. Other drugs tested in different trials failed to demonstrate any benefit. Disappointing results of numerous trials testing the efficacy of various drugs indicate that HCC has low sensitivity to chemotherapy that is in great part caused by multidrug resistance. Immunotherapy for HCC is a new challenging treatment option and involves immune checkpoint inhibitors/antibody-based therapy and peptide-based vaccines. Another challenging approach is microRNA-based therapy that involves two strategies. The first aims to inhibit oncogenic miRNAs by using miRNA antagonists and the second strategy is miRNA replacement, which involves the reintroduction of a tumor-suppressor miRNA mimetic to restore a loss of function.