Structure-activity relationship and cytotoxicity of the new thiosemicarbazide derivatives and their Cu(II) complexes against prostate and melanoma cancer cells.

In this study, eighteen new ligands (B1-B18) containing a thiosemicarbazide core were synthesized and characterized in terms of physicochemical properties, molecular docking and in vitro biological activity. The structures of eleven ligands were investigated using X-Ray diffraction and Hirschfeld Surface analysis. To study the structure-activity relationship, the organic ligands contained pyridin-2-ylmethyl, pyridin-3-ylmethyl or pyridin-4-ylmethyl moieties and various substituents. Their pharmakokinetic profiles and molecular docking results suggest high potential as new drug candidates. The complexing ability of the selected organic ligands was also evaluated, yielding five new Cu(II) complexes (Cu(B1)Cl2, Cu(B4)Cl2, Cu(B10)Cl2, Cu(B17)Cl2, Cu(B18)Cl2). The obtained results suggest the formation of the polymeric structures. All organic ligands and Cu(II) complexes were tested for anticancer activity against prostate and melanoma cancer cells (PC-3, DU-145, LNCaP, A375, G-361, SK-MEL-28) and normal fibroblasts (BJ), as well as antimicrobial activity against six selected bateria strains. Among B1-B18 compounds, B3, B5, B9, B10, B12 and B14 exhibited cytotoxic activity. The studied Cu(II) complexes were in general more active, with Cu(B1)Cl2 exhibiting antincancer activity agains all three prostate cancer cells and Cu(B10)Cl2 reaching the IC50 value equal to 88 µM against G-361 melanoma cells. Several compounds also exhibited antimicrobial activity against gram-positive and gram-negative bacteria. It was found that the type of specific substituents, especially the presence of -chloro and -dichloro substituents had a greated impact on the cytotoxicity than the position of the nitrogen atom in the pyridylacetyl moiety.

2,4-dinitrophenol enhances cisplatin and etoposide cytotoxic activity on prostate cancer cell line.

INTRODUCTION AND OBJECTIVE: Including additional compounds that disturb the energy metabolism of cancer cells in advanced cancer therapy regimens may be an approach to overcome the problem of drug resistance and the therapeutic effectiveness of classic chemotherapeutics. One of the compounds that decouple oxidative phosphorylation, and thus alter the activity of energy-producing pathways, is 2,4-DNP (2,4- dinitrophenol). OBJECTIVE: The aim of the study was to assess the ability of the 2,4-DNP to sensitize prostate cancer cells to the action of cisplatin and etoposide, or to intensify their action. MATERIAL AND METHODS: The research was carried out on three prostate cancer cell lines (LNCaP, PC-3, DU-145. To assess the effect of cisplatin or etoposide with 2,4-DNP on prostate cancer cells, MTT assay, analysis of the cell cycle and apoptosis detection was performed. Oxidative stress was investigated by CellRox fluorescence staining and expression of genes related to antioxidant defence. In addition, analysis was conducted of the expression of genes related to cell cycle inhibition, transporters associated with multi-drug resistance and DNA repair. RESULTS: The study showed that the simultaneous incubation of 2,4-DNP with cisplatin or etoposide enhances the cytotoxic effect of the chemotherapeutic agent only in LNCaP cells (oxidative phenotype). CONCLUSIONS: The enhanced cytotoxic effect of chemotherapeutics by 2,4-DNP may be the result of disturbed redox balance, reduced ability of cells to repair DNA, and the oxidative metabolic phenotype of prostate cancer cells.

Heterogeneous Cellular Response of Primary and Metastatic Human Gastric Adenocarcinoma Cell Lines to Magnoflorine and Its Additive Interaction with Docetaxel.

Gastric cancer is the most common cancer and remains the leading cause of cancer death worldwide. In this study, the anticancer action of magnoflorine isolated via counter-current chromatography from the methanolic extract of Berberis vulgaris root against gastric cancer in models of primary ACC-201 and AGS and metastatic MKN-74 and NCI-N87 cell lines was analyzed. Cell viability and proliferation were tested through the use of MTT and BrdU tests, respectively. Cell cycle progression and apoptosis were evaluated using flow cytometry. The interaction of magnoflorine and docetaxel has been examined through isobolographic analysis. Moreover, potential toxicity was verified in zebrafish in an in vivo model. Gastric cancer cell lines revealed different responses to magnoflorine treatment with regard to viability/proliferation, apoptosis induction and cell cycle inhibition without any undesirable changes in the development of larval zebrafish at the tested concentrations. What is more, magnoflorine in combination with docetaxel produced an additive pharmacological interaction in all studied gastric cancer cell lines, which may suggest a complementary mechanism of action of both compounds. Taken together, these findings provide a foundation for the possibility of magnoflorine as a potential therapeutic approach for gastric cancer and merits further investigation, which may pave the way for clinical uses of magnoflorine.

Novel phenoxyacetylthiosemicarbazide derivatives as novel ligands in cancer diseases.

Numerous epidemiological studies report an increased risk of developing prostate cancer in patients with melanoma and an increased risk of developing melanoma in patients with prostate cancer. Based on our previous studies demonstrating the high anticancer activity of thiosemicarbazides with a phenoxy moiety, we designed nineteen phenoxyacetylthiosemicarbazide derivatives and four of them acting as potential dual-ligands for both cancers. All of the compounds were characterized by their melting points and 1H, 13C NMR and IR spectra. For selected compounds, X-ray investigations were carried out to confirm the synthesis pathway, identify the tautomeric form and intra- and intermolecular interaction in the crystalline state. The conformational preferences and electronic structure of molecules were investigated by theoretical calculation method. Lipophilicity of compounds (log kw) was determined using isocratic reversed phase/high pressure liquid chromatography RP-18. For the obtained compounds, in vitro tests were carried out on four melanoma cell lines (A375, G-361, SK-MEL2, SK-MEL28), four prostate cancer cell lines (PC-3, DU-145, LNCaP, VcaP) and a normal human fibroblast cell line (BJ). The most active compounds turned out to be F6. Cell cycle analysis, apoptosis detection, CellROX staining and mitochondrial membrane potential analysis were performed for the most sensitive cancer cells treated with most active compounds. DSC analysis was additionally performed for selected compounds to determine their purity, compatibility, and thermal stability. The process of prooxidation was proposed as a potential mechanism of anticancer activity.

Disruption of mitochondrial function augments the radiosensitivity of prostate cancer cell lines.

INTRODUCTION: Ionizing radiation is one of the most widely used therapeutic methods in the treatment of prostate cancer, but the problem is developing radioresistance of the tumour. There is evidence that metabolic reprogramming in cancer is one of the major contributors to radioresistance and mitochondria play a crucial role in this process. OBJECTIVE: The aim of the study was to assess the influence of oxidative phosphorylation uncoupling to radiosensitivity of prostate cancer cells differing in metabolic phenotype. MATERIAL AND METHODS: LNCaP, PC-3 and DU-145 cells were exposed to X-rays and simultaneously treated with 2,4-dinitrophenol (2,4-DNP). The radiosensitive of cell lines was determined by cell clonogenic assay and cell cycle analysis. The cytotoxic effect was evaluated with MTT and CVS (Crystal violet staining) assay, apoptosis detection and cell cycle analysis. The phenotype of the cells was determined by glucose uptake and lactate release, ATP level measurement as well as basal reactive oxygen species level and mRNA expression of genes related to oxidative stress defence. RESULTS: The synergistic effect of 2,4-dinitrophenol and X-ray was observed only in the case of the LNCaP cell line. CONCLUSIONS: Phenotypic analysis indicates that this may be due to the highest dependence of these cells on oxidative phosphorylation and sensitivity to disruption of their redox status.

Cardioprotective Effect of Centaurea castriferrei Borbás & Waisb Extract against Doxorubicin-Induced Cardiotoxicity in H9c2 Cells.

Doxorubicin (DOX) is one of the most used chemotherapeutic agents in the treatment of various types of cancer. However, a continual problem that is associated with its application in therapeutic regimens is the development of dose-dependent cardiotoxicity. The progression of this process is associated with a range of different mechanisms, but especially with the high level of oxidative stress. The aim of the study was to evaluate the effects of the water and methanol-water extracts from the plant Centaurea castriferrei (CAS) obtained by the ultrasound-assisted extraction method on the DOX-induced cardiotoxicity in the rat embryonic cardiomyocyte cell line H9c2. The H9c2 cells were treated for 48 h with the DOX and water or methanol-water extracts, or a combination (DOX + CAS H2O/CAS MeOH). The MTT assay, cell cycle analysis, and apoptosis detection revealed that both the tested extracts significantly abolished the cytotoxic effect caused by DOX. Moreover, the detection of oxidative stress by the CellROX reagent, the evaluation of the number of AP sites, and the expressions of the genes related to the oxidative stress defense showed substantial reductions in the oxidative stress levels in the H9c2 cells treated with the combination of DOX and CAS H2O/CAS MeOH compared with the DOX administered alone. The tested extracts did not affect the cytotoxic effect of DOX on the MCF-7 breast cancer cell line. The obtained results constitute the basis for further research in the context of the application of C. castriferrei extracts as adjuvants in the therapy regiments of cancer patients treated with DOX.

2,4-Dinitrophenol as an Uncoupler Augments the Anthracyclines Toxicity against Prostate Cancer Cells.

One of the strategies for the treatment of advanced cancer diseases is targeting the energy metabolism of the cancer cells. The compound 2,4-DNP (2,4-dinitrophenol) disrupts the cell energy metabolism through the ability to decouple oxidative phosphorylation. The aim of the study was to determine the ability of 2,4-DNP to sensitize prostate cancer cells with different metabolic phenotypes to the action of known anthracyclines (doxorubicin and epirubicin). The synergistic effect of the anthracyclines and 2,4-DNP was determined using an MTT assay, apoptosis detection and a cell cycle analysis. The present of oxidative stress in cancer cells was assessed by CellROX, the level of cellular thiols and DNA oxidative damage. The study revealed that the incubation of LNCaP prostate cancer cells (oxidative phenotype) with epirubicin and doxorubicin simultaneously with 2,4-DNP showed the presence of a synergistic effect for both the cytostatics. Moreover, it contributes to the increased induction of oxidative stress, which results in a reduced level of cellular thiols and an increased number of AP sites in the DNA. The synergistic activity may consist of an inhibition of ATP synthesis and the simultaneous production of toxic amounts of ROS, destroying the mitochondria. Additionally, the sensitivity of the LNCaP cell line to the anthracyclines is relatively higher compared to the other two (PC-3, DU-145).

Phytochemical Analysis and Anti-Cancer Properties of Extracts of Centaurea castriferrei Borbás & Waisb Genus of Centaurea L.

The Centaurea L. (Asteraceae) genus includes many plant species with therapeutic properties. Centaurea castriferrei Borbás & Waisb is one of the least known and least described plants of this genus. The aim of the study was the phytochemical analysis of water and methanol-water extracts (7:3 v/v) obtained from the aerial parts of the plant as well as evaluation of their anticancer activity. Quantitative determinations of phenolic compounds and flavonoids were performed, and the antioxidant potential was measured using the CUPRAC method. The RP-HPLC/DAD analysis and HPLC-ESI-QTOF-MS mass spectroscopy were performed, to determine the extracts' composition. The antiproliferative activity of the obtained extracts was tested in thirteen cancer cell lines and normal skin fibroblasts using MTT test. Regardless of the extraction method and the extractant used, similar cytotoxicity of the extracts on most cancer cell lines was observed. However, the methanol-water extracts (7:3 v/v) contained significantly more phenolic compounds and flavonoids as well as showing stronger antioxidant properties in comparison to water extracts. Centaurea castriferrei Borbás & Waisb is a rich source of apigenin and its derivatives. In all tested extracts, chlorogenic acid and centaurein were also identified. In vitro research revealed that this plant may be a potential source of compounds with anticancer activity.

Phytochemical Profile and Biological Activity of the Ethanolic Extract from the Aerial Part of Crocus alatavicus Regel & Semen Growing Wildly in Southern Kazakhstan.

The composition of the ethanolic extract from the aerial parts of Crocus alatavicus Regel & Semen from southern Kazakhstan spontaneous flora was analyzed together with the determination of its antibacterial, antifungal, antiviral and anticancer activity. The phytochemical profile analysis by high-performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (HPLC/ESI-QTOF-MS) revealed the presence of multiple kaempferol derivatives. High-performance reverse-phase liquid chromatography combined with a photodiode-array detection (RP-HPLC/PDA) found that kaempferol 3-O-dihexoside and kaempferol 3-O-acyltetrahexoside accounted for 70.5% of the kaempferol derivatives. The minimum inhibitory concentration (MIC) values of the extract for all the tested reference microorganisms were high, reaching 10 mg/mL for yeasts and 20 mg/mL for bacteria. In contrast, antiviral activity was observed at 2 mg/mL, resulting in the inhibition of the HSV-1-induced cytopathic effect and the reduction in virus infectious titer by 1.96 log, as well as the viral load by 0.85 log. Among the tested prostate cancer cell lines, significant cytotoxic activity of the extract was noted only on the LNCaP cell line, with an IC50 value of 1.95 mg/mL. The LNCaP cell line treated with 2 mg/mL of the extract showed a noticeably reduced number of spindle-shaped cells with longer cellular projections, a significant increase in the peak corresponding to the population of apoptotic cells in the sub-G1 phase and a decreased intracellular glutathione (GSH) level, suggesting the prooxidative properties of the extract. The obtained data provide novel information about the flavonoids present in the aerial part of C. alatavicus and suggest its potential application as a source of the compounds active against HSV-1 and metastatic, androgen-sensitive prostate cancer.

Targeting Energy Metabolism in Cancer Treatment.

Cancer is the second most common cause of death worldwide after cardiovascular diseases. The development of molecular and biochemical techniques has expanded the knowledge of changes occurring in specific metabolic pathways of cancer cells. Increased aerobic glycolysis, the promotion of anaplerotic responses, and especially the dependence of cells on glutamine and fatty acid metabolism have become subjects of study. Despite many cancer treatment strategies, many patients with neoplastic diseases cannot be completely cured due to the development of resistance in cancer cells to currently used therapeutic approaches. It is now becoming a priority to develop new treatment strategies that are highly effective and have few side effects. In this review, we present the current knowledge of the enzymes involved in the different steps of glycolysis, the Krebs cycle, and the pentose phosphate pathway, and possible targeted therapies. The review also focuses on presenting the differences between cancer cells and normal cells in terms of metabolic phenotype. Knowledge of cancer cell metabolism is constantly evolving, and further research is needed to develop new strategies for anti-cancer therapies.

Hyaluronic Acid as a Modern Approach in Anticancer Therapy-Review.

Hyaluronic acid (HA) is a linear polysaccharide and crucial component of the extracellular matrix (ECM), maintaining tissue hydration and tension. Moreover, HA contributes to embryonic development, healing, inflammation, and cancerogenesis. This review summarizes new research on the metabolism and interactions of HA with its binding proteins, known as hyaladherins (CD44, RHAMM), revealing the molecular basis for its distinct biological function in the development of cancer. The presence of HA on the surface of tumor cells is a sign of an adverse prognosis. The involvement of HA in malignancy has been extensively investigated using cancer-free naked mole rats as a model. The HA metabolic components are examined for their potential impact on promoting or inhibiting tumor formation, proliferation, invasion, and metastatic spread. High molecular weight HA is associated with homeostasis and protective action due to its ability to preserve tissue integrity. In contrast, low molecular weight HA indicates a pathological condition in the tissue and plays a role in pro-oncogenic activity. A systematic approach might uncover processes related to cancer growth, establish novel prognostic indicators, and identify potential targets for treatment action.

Deacetylation of Transcription Factors in Carcinogenesis.

Reversible Nε-lysine acetylation/deacetylation is one of the most common post-translational modifications (PTM) of histones and non-histone proteins that is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). This epigenetic process is highly involved in carcinogenesis, affecting histone and non-histone proteins' properties and their biological functions. Some of the transcription factors, including tumor suppressors and oncoproteins, undergo this modification altering different cell signaling pathways. HDACs deacetylate their targets, which leads to either the upregulation or downregulation of proteins involved in the regulation of cell cycle and apoptosis, ultimately influencing tumor growth, invasion, and drug resistance. Therefore, epigenetic modifications are of great clinical importance and may constitute a new therapeutic target in cancer treatment. This review is aimed to present the significance of HDACs in carcinogenesis through their influence on functions of transcription factors, and therefore regulation of different signaling pathways, cancer progression, and metastasis.

Palmatine, a Bioactive Protoberberine Alkaloid Isolated from Berberis cretica, Inhibits the Growth of Human Estrogen Receptor-Positive Breast Cancer Cells and Acts Synergistically and Additively with Doxorubicin.

Palmatine (PLT) is a natural isoquinoline alkaloid that belongs to the class of protoberberines and exhibits a wide spectrum of pharmacological and biological properties, including anti-cancer activity. The aim of our study was to isolate PLT from the roots of Berberis cretica and investigate its cytotoxic and anti-proliferative effects in vitro alone and in combination with doxorubicine (DOX) using human ER+/HER2- breast cancer cell lines. The alkaloid was purified by column chromatography filled with silica gel NP and Sephadex LH-20 resin developed in the mixture of methanol: water (50:50 v/v) that provided high-purity alkaloid for bioactivity studies. The purity of the alkaloid was confirmed by high resolution mass measurement and MS/MS fragmentation analysis in the HPLC-ESI-QTOF-MS/MS-based analysis. It was found that PLT treatment inhibited the viability and proliferation of breast cancer cells in a dose-dependent manner as demonstrated by MTT and BrdU assays. PLT showed a quite similar growth inhibition on breast cancer cells with IC50 values ranging from 5.126 to 5.805 µg/mL. In contrast, growth of normal human breast epithelial cells was not affected by PLT. The growth inhibitory activity of PLT was related to the induction of apoptosis, as determined by Annexin V/PI staining. Moreover, PLT sensitized breast cancer cells to DOX. Isobolographic analysis revealed synergistic and additive interactions between studied agents. Our studies suggest that PLT can be a potential candidate agent for preventing and treating breast cancer.

The Mitochondria-Independent Cytotoxic Effect of Leflunomide on RPMI-8226 Multiple Myeloma Cell Line.

Leflunomide, an anti-inflammatory agent, has been shown to be effective in multiple myeloma (MM) treatment; however, the mechanism of this phenomenon has not been fully elucidated. The aim of the study was to assess the role of mitochondria and dihydroorotate dehydrogenase (DHODH) inhibition in the cytotoxicity of leflunomide in relation to the MM cell line RPMI 8226. The cytotoxic effect of teriflunomide-an active metabolite of leflunomide-was determined using MTT assay, apoptosis detection, and cell cycle analysis. To evaluate DHODH-dependent toxicity, the cultures treated with teriflunomide were supplemented with uridine. Additionally, the level of cellular thiols as oxidative stress symptom was measured as well as mitochondrial membrane potential and protein tyrosine kinases (PTK) activity. The localization of the compound in cell compartments was examined using HPLC method. Teriflunomide cytotoxicity was not abolished in uridine presence. Observed apoptosis occurred in a mitochondria-independent manner, there was also no decrease in cellular thiols level. Teriflunomide arrested cell cycle in the G2/M phase which is not typical for DHODH deficiency. PTK activity was decreased only at the highest drug concentration. Interestingly, teriflunomide was not detected in the mitochondria. The aforementioned results indicate DHODH- and mitochondria-independent mechanism of leflunomide toxicity against RPMI 8226 cell line.

Influence of Complexation of Thiosemicarbazone Derivatives with Cu (II) Ions on Their Antitumor Activity against Melanoma Cells.

A series of thiosemicarbazone derivatives was prepared and their anti-tumor activity in vitro was tested. The X-ray investigation performed for compounds T2, T3 and T5 confirmed the synthesis pathway and assumed molecular structures of analyzed thiosemicarbazones. The conformational preferences of the thiosemicarbazone system were characterized using theoretical calculations by AM1 method. Selected compounds were converted into complexes of Cu (II) ions. The effect of complexing on anti-tumor activity has been investigated. The copper(II) complexes, with Schiff bases T1, T10, T12, T13, and T16 have been synthesized and characterized by chemical and elemental analysis, FTIR spectroscopy and TGA method. Thermal properties of coordination compounds were studied using TG-DTG techniques under dry air atmosphere. G361, A375, and SK-MEL-28 human melanoma cells and BJ human normal fibroblast cells were treated with tested compounds and their cytotoxicity was evaluated with MTT test. The compounds with the most promising anti-tumour activity were then selected and their cytotoxicity was verified with cell cycle analysis and apoptosis/necrosis detection. Additionally, DNA damages in the form of a basic sites presence and the expression of oxidative stress and DNA damage response genes were evaluated. The obtained results indicate that complexation of thiosemicarbazone derivatives with Cu (II) ions improves their antitumor activity against melanoma cells. The observed cytotoxic effect is associated with DNA damage and G2/M phase of cell cycle arrest as well as disorders of the antioxidant enzymes expression.

Apigenin and Hesperidin Downregulate DNA Repair Genes in MCF-7 Breast Cancer Cells and Augment Doxorubicin Toxicity.

A number of studies have confirmed anti-tumor activity of flavonoids and their ability to enhance the effectiveness of classical anticancer drugs. The mechanism of this phenomenon is difficult to explain because of the ambivalent nature of these compounds. Many therapeutic properties of these compounds are attributed to their antioxidant activity; however, it is known that they can act as oxidants. The aim of this study was to assess the influence of apigenin and hesperidin on MCF-7 breast cancer cells with doxorubicin. The cytotoxic effect was determined using an MTT test and cell cycle analysis. To evaluate the possible interaction mechanism, reduced glutathione levels, as well as the DNA oxidative damage and the double strand breaks, were evaluated. Additionally, mRNA expression of genes related to DNA repair was assessed. It was demonstrated that flavonoids intensified the cytotoxic effect of doxorubicin despite flavonoids reduced oxidative damage caused by the drug. At the same time, the number of double strand breaks significantly increased and expression of tested genes was downregulated. In conclusion, both apigenin and hesperidin enhance the cytotoxic effects of doxorubicin on breast cancer cells, and this phenomenon occurs regardless of oxidative stress but is accompanied by disorders of DNA damage response mechanisms.

Cytotoxic activity of methanolic fractions of different Marrubium spp. against melanoma cells is independent of antioxidant activity and total phenolic content.

The Marrubium genus (horehound) has proved to be an abundant source of biologically active compounds, but there is little knowledge about its potential anticancer activity. Moreover, some Marrubium species have not been the subject of study in this regard. In this study, we performed comparative analysis of phenolic acid (PhA) content and total phenolic content in fractions obtained from methanolic extracts of Marrubium vulgare L. (common horehound), Marrubium cylleneum Boiss. & Heldr. and Marrubium friwaldskyanum Boiss herbs. We examined the cytotoxicity of these fractions against a human melanoma cancer cell line (A375) and normal human skin fibroblasts (BJ) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test, cell cycle analysis and real-time monitoring of cell viability. We detected caffeic, p-coumaric, ferulic and gentisic acids among the PhAs. Although the extracts obtained demonstrated low total phenolic content and did not show significant antioxidative properties, the nonhydrolyzed PhA fraction exhibited cytotoxic activity against a human melanoma cancer cell line, without affecting normal fibroblasts. Both acidic and alkaline hydrolysis abolished this activity, indicating that the esterified forms of phenolic compounds caused the observed cytotoxic effects. Further investigation of these compounds may facilitate the development of novel drugs for cancer treatment.

FSHR Trans-Activation and Oligomerization.

Follicle stimulating hormone (FSH) plays a key role in human reproduction through, among others, induction of spermatogenesis in men and production of estrogen in women. The function FSH is performed upon binding to its cognate receptor-follicle-stimulating hormone receptor (FSHR) expressed on the surface of target cells (granulosa and Sertoli cells). FSHR belongs to the family of G protein-coupled receptors (GPCRs), a family of receptors distinguished by the presence of various signaling pathway activation as well as formation of cross-talking aggregates. Until recently, it was claimed that the FSHR occurred naturally as a monomer, however, the crystal structure as well as experimental evidence have shown that FSHR both self-associates and forms heterodimers with the luteinizing hormone/chorionic gonadotropin receptor-LHCGR. The tremendous gain of knowledge is also visible on the subject of receptor activation. It was once thought that activation occurs only as a result of ligand binding to a particular receptor, however there is mounting evidence of trans-activation as well as biased signaling between GPCRs. Herein, we describe the mechanisms of aforementioned phenomena as well as briefly describe important experiments that contributed to their better understanding.