Anticancer activity of new molecular hybrids combining 1,4-naphthalenedione motif with phosphonic acid moiety in hepatocellular carcinoma HepG2 cells.

Structural motifs found in naturally occurring compounds are frequently used by researchers to develop novel synthetic drug candidates. Some of these new agents are hybrid molecules which are designed through a concept of combining more than one functional element. In this report, anticancer activity of new synthetic molecular hybrids, substituted 3-diethoxyphosphorylnaphtho[2,3-b]furan-4,9-diones and 3-diethoxyphosphorylbenzo[f]indole-4,9-diones, which integrate natural 1,4-naphtalenedione scaffold, present in several anticancer agents, with pharmacophoric phosphonate moiety, were tested against hepatocellular cell line HepG2. Cytotoxicity was examined using MTT assay. Two most potent compounds, furandione 8a and benzoindoldione 12a, which reduced the number of viable HepG2 cells with the IC50 values of 4.13 µM and 5.9 µM, respectively, were selected for further research. These compounds decreased the mRNA expression levels of several genes: Bcl-2, angiogenic vascular endothelial growth factor (VEGF), c-Fos, caspase-8 and increased the expression of Bax, caspase-3 and -9, c-Jun, p21, p53, as determined by quantitative real-time PCR. The ability of these compounds to induce apoptosis and DNA damage was studied by flow cytometry. The obtained data showed that the new compounds inhibited cell viability by increasing apoptosis and decreasing angiogenesis. Compound 8a was a much stronger apoptosis inducer as compared with 12a and strongly activated the intrinsic pathway of apoptosis, associated with the loss of mitochondrial membrane potential and changes in Bax/Bcl-2 ratio. These findings show that the synthetic hybrids combining 1,4-naphthalenedione system and phosphonic acid moiety display potential to be further explored in the development of new anticancer agents.

Anticancer properties of new synthetic hybrid molecules combining naphtho[2,3-b]furan-4,9-dione or benzo[f]indole-4,9-dione motif with phosphonate subunit.

In this paper we report an efficient and general synthesis of substituted 3-diethoxyphosphorylnaphtho [2,3-b]furan-4,9-diones and 3-diethoxyphosphorylbenzo [f]indole-4,9-diones which integrate the natural 1,4-naphtalenedione scaffold, present in several anticancer agents with the phosphonate moiety. The cytotoxicity of such hybrid molecules was tested against two leukemia cell lines, HL-60 and NALM-6 and against a breast adenocarcinoma MCF-7 cell line. Selected compounds were also tested on normal human cells: HUVEC and MCF-10A. In general, naphthofuran-4,9-diones showed much higher cytotoxic activity (IC50 values below 10 µM) than benzoindole-4,9-diones. The most promising 2-(2-chlorophenyl)-3-diethoxyphosphorylnaphtho [2,3-b]furan-4,9-dione, with the highest cytotoxic activity in the MTT test, was chosen for further evaluation of its anticancer potential. This compound, tested on HL-60 and MCF-7 cells inhibited cell proliferation, generated DNA damage and induced apoptosis. The suggested mechanism of its cytotoxic activity was the generation of intracellular reactive oxygen species and the induction of mitochondrial membrane potential dissipation.

Anticancer activity and radiosensitization effect of methyleneisoxazolidin-5-ones in hepatocellular carcinoma HepG2 cells.

Parthenolide (PTL), a well-known sesquiterpene lactone of natural origin with α,ß-unsaturated carbonyl structure, has proven to show promising anti-cancer properties. In this report, anti-proliferative potential of two synthetic methyleneisoxazolidin-5-ones, MZ-6 and MZ-14, with the same structural motif, has been investigated in human hepatoma HepG2 cells. The effects on apoptosis induction and DNA damage were evaluated. All compounds decreased the number of live cells and increased the number of late apoptotic cells. However, only MZ-14 was able to induce DNA damage. Both synthetic compounds increased intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential changes at the same level as PTL. Additionally, cell survival was analyzed after a combined treatment, in which HepG2 cells were preincubated for 24 h with MZ-6, MZ-14 or PTL and irradiated with different doses of X-rays. The inhibition of cell survival was assessed by the clonogenic assay. We have shown that the clone formation was strongly inhibited by the combined treatment. The synergistic effect was observed for all three compounds but MZ-6 was significantly more effective. It is interesting to note that in HepG2 cells MZ-6 was the least cytotoxic of the tested compounds, did not induce DNA damage and was less active than the others in the clonogenic cell survival assay. It seems advantages from the point of view of the further in vivo studies that the compound with the lowest cytotoxic activity showed the strongest sensitizing effect.

Anticancer properties of novel 4-methylene-1,2-diphenylpyrazolidin-3-ones.

The limited success of the currently used antitumor therapies is the driving force for organic chemists to seek new lead structures with anticancer potential. Two α-methylene-γ-lactams with an additional nitrogen atom in the lactam ring, 5-vinyl-1,2-diphenyl-4-methylenepyrazolidin-3-one (2a) and 5-phenyl-1,2-diphenyl-4-methylenepyrazolidin-3-one (2b) have been synthesized. Their anticancer activity was assessed in MCF-7 cells. Both compounds inhibited cell proliferation and induced DNA damage and apoptosis, with 2a being the more potent analog. Synergistic effects of 2a used in combination with known anticancer drugs, 5-fluorouracil, taxol, and oxaliplatin were evaluated. Compound 2a significantly enhanced the antitumor action of oxaliplatin and 5-fluorouracil, but not taxol.

Combined effects of anticancer drugs and new synthetic α-methylene-δ-lactones on MCF-7 cells.

The search for novel drug candidates is a priority goal for cancer therapy. Natural products isolated from plants are often used as valuable leads for the synthesis of analogs with simpler structure. Two synthetic α-methylene-δ-lactones with chroman-2-one skeleton, designated DL-3 and DL-5, exhibiting strong cytotoxic activity against several cancer cell lines, have been tested alone and in combination with well-known anticancer drugs, 5-fluorouracil, oxaliplatin, and taxol, in breast cancer MCF-7 cells. Parthenolide, a plant-derived α-methylene-γ-lactone, was used as a positive control. The effects on cell proliferation, DNA damage, and apoptosis induction were evaluated. Neither of the tested compounds significantly enhanced the effects produced by taxol, but a strong synergistic effect was observed with 5-fluorouracil and oxaliplatin. Only small differences between the actions of both α-methylene-δ-lactones were found. The synergistic effects produced by these compounds in MCF-7 cells were stronger as compared with parthenolide. Our findings show that simple and easy-to-obtain synthetic compounds with α-methylene-δ-lactone motif can potentiate the efficiency of anticancer drugs.

The role of oxidative stress in anticancer activity of sesquiterpene lactones.

Sesquiterpene lactones (SLs) are plant-derived compounds that are abundant in plants of the Asteraceae family and posses a broad spectrum of biological activities, ranging from anti-inflammatory, phytotoxic, antibacterial, and antifungal to cytotoxic/anticancer. In recent years, anticancer properties of these compounds and molecular mechanisms of their action have been studied extensively on numerous cell lines and also on experimental animals. SLs have been shown to disrupt cellular redox balance and induce oxidative stress in cancer cells. Oxidative stress is associated with increased production of reactive oxygen species (ROS) which in turn can promote many aspects of cancer development and progression. On the other hand, ROS, which initiate apoptosis via the mitochondrial-dependent pathway, can also be used to kill cancer cells, if they can be generated in cancer. One of the most important regulators of the redox equilibrium in the cells is reduced glutathione (GSH). In cancer cells, GSH levels are higher than in normal cells. Therefore, SL can induce apoptosis of cancer cells by decreasing intracellular GSH levels. The use of SL which can affect intracellular redox signaling pathways can be considered an interesting approach for cancer treatment. In this review, we give a brief description of the mechanisms and pathways involved in oxidative stress-induced anticancer activity of SL.

Design, synthesis and cytotoxic evaluation of 4-methylidenepyrazolidin-3-ones.

Three series of new 4-methylidenepyrazolidin-3-ones with various substitution patterns were synthesized and tested for the cytotoxic activity against two human leukemia cell lines NALM-6 and HL-60 as well as MCF-7 breast cancer cell line. Several obtained methylidenepyrazolidinones exhibited high cytotoxic activity with IC50 values below 10 µM, mainly against HL-60 leukemia cell line and two of them, 18d,e, displayed IC50 ≤ 5 µM, against all tested cell lines. Structure-activity relationship studies revealed that the presence of phenyl substituents on both ring nitrogen atoms and vinyl or phenyl substituents in position 5 are crucial for high activity. Selected methylidenepyrazolidinones were also tested on normal human umbilical vein endothelial cells (HUVEC) and pyrazolidinone 18a was found to be 5-fold more toxic against HL-60 than normal cells.

Proteomic analysis of proteins engaged in α-methylene-δ-lactone cytotoxic effects in hormone-independent breast cancer MDA-MB-231 cells.

A simple synthetic α-methylene-δ-lactone, 1-isopropyl-2-methylene-1,2-dihydrobenzochromen-3-one, designated DL-3, was shown previously to induce apoptosis and significantly suppress cell metastatic potential in MDA-MB-231 breast cancer cells. The mechanisms through which DL-3 exerts its effects are poorly understood. The purpose of this study was to investigate the protein expression profiles in MDA-MB-231 cells exposed to the DL-3 treatment. Using 2D differential gel electrophoresis, a set of eight differentially expressed proteins (spot intensities which showed ≥1.25-fold change and statistical significance, p < 0.05, between the control and DL-3-treated group) were found and successfully identified by mass spectrometry (MALDI-TOF/MS). The proteomic results revealed that the presence of DL-3 in MDA-MB-231 cells led to the differential regulation of some proteins that are involved in the cell cycle progression, apoptosis, cytokinesis, modulation of transcription, cellular signaling, and vesicular trafficking. The function of other identified proteins is still unknown. Therefore, our data indicate new directions for the further studies of the pathways engaged in the anticancer action exerted by α-methylene-δ-lactones in cancer cells.

α-Methylene-γ-lactones as a novel class of anti-leukemic agents.

Natural products are important leads in drug discovery. In recent years, the anti-leukemic properties of natural compounds isolated from plants, containing an α-Methylene-γ-lactones skeleton, have attracted a lot of attention. Extensive research has been carried out to characterize their molecular mechanisms of action and potential chemotherapeutic application in different types of cancer, including leukemias. Sesquiterpene lactones, a group of α-Methylene-γ-lactones are plant-derived compounds, mostly of the Compositae family, used in traditional medicine especially for the treatment of inflammation. However, they exhibit a broad spectrum of other biological effects, including cytotoxic, anti-bacterial, anti-helminthic, and anti-tumor activity. Recently, a sesquiterpene lactone, parthenolide, and several other compounds containing an α-methylene-γ-lactone skeleton have become topics of interest as potential antileukemic agents. The recent research emphasizes their selective activity against leukemia cells while the normal hematopoietic cells remain unaffected. In this review, we give a brief description of natural α-Methylene-γ-lactones isolated from plants and their derivates with minor chemical modifications that possess anti-leukemic activity. We also discuss molecular mechanisms of action of these compounds, in particular, their selectivity against leukemia cells.

Combined effect of parthenolide and various anti-cancer drugs or anticancer candidate substances on malignant cells in vitro and in vivo.

Despite the enormous advances in the development of new drugs, the efficacy of current antitumor therapies is still quite limited, most likely because of the high degree of cancer heterogeneity and cell signaling complexity. As a single drug does not necessarily eradicate the cancer, the use of drug combinations has been proposed, and numerous studies have already been conducted to examine the efficacy of this strategy. In the last decade, parthenolide, a plant-derived sesquiterpene lactone, was extensively studied and its potential to inhibit cancer cell growth in vitro was well documented. More recently, antitumor effects exerted by parthenolide in combination with various drugs routinely used in cancer treatment have been investigated in several laboratories. In this article studies that are underway in an attempt to improve the anticancer efficacy of chemotherapies through combination strategies involving parthenolide are summarized.

Immunomodulatory effects of endogenous and synthetic peptides activating opioid receptors.

The main role of endogenous opioid peptides is the modulation of pain. Opioid peptides exert their analgesic activity by binding to the opioid receptors distributed widely in the central nervous system (CNS). However, opioid receptors are also found on tissues and organs outside the CNS, including the cells of the immune system, indicating that opioids are capable of exerting additional effects in periphery. Morphine, which is a gold standard in the treatment of chronic pain, is well-known for its immunosuppressive effects. Much less is known about the immunomodulatory effects exerted by endogenous (enkephalins, endorphins, dynorphins and endomorphins) and synthetic peptides activating opioid receptors. In this review we tried to summarize opioid peptide-mediated modulation of immune cell functions which can be stimulatory as well as inhibitory.

Anticancer Activity of New Synthetic α-Methylene-δ-Lactones on Two Breast Cancer Cell Lines.

Natural products are important leads in drug discovery. The search for effective plant-derived anticancer agents or their synthetic analogues has continued to be of interest to biologists and chemists for a long time. In this report, cytotoxicity and anticancer activity of new synthetic α-methylene-δ-lactones was tested against two breast cancer cell lines, invasive, hormone-independent MDA-MB-231 and hormone-dependent MCF-7. Cytotoxicity was examined using MTT assay. The ability to induce apoptosis and changes in mitochondrial membrane potential was studied by flow cytometry. The expression levels of pro- and anti-apoptotic genes were determined by quantitative real-time PCR. Cancer cell migration and invasion were assessed by wound healing and Matrigel assays. Additionally, secretion of proteins associated with invasiveness, metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) was investigated using commercial ELISA kits and MMP-9 activity by gelatin zymography. A natural sesquiterpene lactone, parthenolide, was used as a positive control. Screening results showed all four analogues to be highly cytotoxic. The most potent compound of the series, 1-isopropyl-2-methylene-1,2-dihydrobenzochromen-3-one, designated DL-3, which reduced the number of viable MDA-MB-231 and MCF-7 cells with the IC50 values of 5.3 µM and 3.54 µM, respectively, was selected for further research. DL-3 activated the intrinsic pathway of apoptosis, associated with the loss of mitochondrial membrane potential and changes in Bax/Bcl-2 ratio. DL-3 also inhibited the movement of both types of breast cancer cells. Suppression of cell migration and invasion was the result of the decreased secretion of enzymes responsible for the degradation of the extracellular matrix, MMP-9 and uPA. These findings show that the synthetic α-methylene-δ-lactone, DL-3, displays potential to be further explored in the development of new anticancer agents.

Cyclization in opioid peptides.

Endogenous opioid peptides have been studied extensively as potential therapeutics for the treatment of pain. The major problems of using natural opioid peptides as drug candidates are their poor receptor specificity, metabolic instability and inability to reach the brain after systemic administration. A lot of synthetic efforts have been made to opioid analogs with improved pharmacological properties. One important structural modification leading to such analogs is cyclization of linear sequences. Intramolecular cyclization has been shown to improve biological properties of various bioactive peptides. Cyclization reduces conformational freedom responsible for the simultaneous activation of two or more receptors, increases metabolic stability and lipophilicity which may result in a longer half-life and easier penetration across biological membranes. This review deals with various strategies that have been employed to synthesize cyclic analogs of opioid peptides. Discussed are such bridging bonds as amide and amine linkages, sulfur-containing bonds, including monosulfide, disulfide and dithioether bridges, bismethylene bonds, monosulfide bridges of lanthionine and, finally, carbonyl and guanidine linkages. Opioid affinities and activities of cyclic analogs are given and compared with linear opioid peptides. Analgesic activities of analogs evaluated in the in vivo pain tests are also discussed.

Apoptosis-mediated cytotoxic effects of parthenolide and the new synthetic analog MZ-6 on two breast cancer cell lines.

The search for effective plant-derived anti-cancer agents or their synthetic analogs has continued to gain interest in drug development. The anti-cancer activity of parthenolide (PTL) isolated from Tanacetum parthenium, has been attributed to the presence of α-methylene-γ-lactone skeleton. In the present study we aimed to investigate the anti-cancer potential of a new synthetic compound, 3-isopropyl-2-methyl-4-methyleneisoxazolidin-5-one (MZ-6), with the same as in PTL α-methylene-γ-lactone motif, on two breast cancer cell lines, MCF-7 and MDA-MB-231. For comparison, PTL was included in the study. PTL and MZ-6 reduced the number of viable MCF-7 and MDA-MB-231 cells, with half maximal inhibitory concentration values between 6 and 9 µM. Both compounds dose-dependently inhibited incorporation of [(3)H]thymidine, up-regulated Bax and down regulated Bcl-2 mRNA. The levels of the end product of lipid peroxidation, malondialdehyde, were significantly higher. In MCF-7 cells, MZ-6 induced early apoptosis and cell cycle arrest in G0/G1 phase. The effect produced by MZ-6 was much stronger compared with PTL. In MDA-MB-231 cells, both tested compounds had similar effect and induced mostly late apoptosis. In conclusion, the observed anticancer activity makes MZ-6 an attractive drug candidate and shows that simple analogs of α-methylene-γ-lactones can be good substitutes for more complex structures isolated from plants.

Synthesis and opioid activity of novel 6-ketolevorphanol derivatives.

Novel 6-ketolevorphanol analogs with diverse substitution patterns at ring C were synthesized and their binding affinities at the µ,δ and κ opioid receptors were investigated. The in vitro activity of the new analogs was then evaluated in the functional assay based on the electrically-stimulated contractions of the mouse ileum. It was shown that analogs with Δ7,8 bond had no significant potency at any of the opioid receptor types. In contrast, analogs with the saturated ring C were either potent κ agonist or antagonist depending on the absence or presence of the hydroxyl group in position 14.

The mu-opioid receptor-selective peptide antagonists, antanal-1 and antanal-2, produce anticonvulsant effects in mice.

The activation of the mu-opioid receptors (MOR) in the central nervous system has a proconvulsant effect and seizures are a common side effect of high doses of short acting opioids, like morphine or fentanyl. However, the correct assessment of the role of MOR blockade in the initiation and propagation of epilepsy was hampered by the lack of potent and selective MOR antagonists. In this study we aimed at characterizing the effect of MOR blockade on the seizure threshold in mice using recently developed selective antagonists antanal-1 and antanal-2 and a classical MOR antagonist, ß-funaltrexamine (ß-FNA). The effect of the centrally administered MOR antagonists was characterized in the maximal electroshock seizure threshold (MEST), the 6 Hz psychomotor seizure threshold and the intravenous pentylenetetrazole (PTZ) seizure threshold test in mice. The acute effect of the studied compounds on skeletal muscular strength in mice was quantified in the grip-strength test. Antanal-1 and antanal-2 (30 and 50 nmol/mouse, i.c.v.), but not ß-FNA significantly increased the seizure threshold in the MEST test in mice. In the 6-Hz test, all tested MOR antagonists significantly increased the psychomotor seizure threshold and the most potent anticonvulsant effect was observed for antanal-2 (2, 10 and 30 nmol/mouse, i.c.v.). The i.c.v. administration of ß-FNA (10 and 30 nmol/mouse, i.c.v.), antanal-1 and antanal-2 (both 30, 50 and 100 nmol/mouse, i.c.v.) did not produce any significant effect on PTZ seizure threshold, the generalized clonus or the forelimbs tonus. All tested compounds did not affect muscle strength, as determined in the grip strength test. Our study demonstrated that the novel MOR-selective antagonists antanal-1 and antanal-2 displayed a potent and dose-dependent anticonvulsant action involving non-GABA-ergic, but some other pathways and mechanisms in animal models of epileptic seizures. We suggest that antanals are promising drug templates for future therapeutics, which may be used in the treatment of epilepsy in humans.

Kinetic studies of novel inhibitors of endomorphin degrading enzymes.

Endomorphins (EMs), two endogenous µ-opioid receptor selective ligands, are attractive lead compounds for opioid-based pain management studies. However, these peptides are quickly degraded by peptidases, in particular by dipeptidylpeptidase IV (DPP IV) and aminopeptidase M (APM). Targeting enzymatic degradation is one approach to prolong endomorphin activity. In this study we characterized the action of two new inhibitors of similar to endomorphins structure, Tyr-Pro-Ala-NH(2) (EMDB-2) and Tyr-Pro-Ala-OH (EMDB-3), which were designed earlier in our laboratory. The presented data give evidence that EMDB-2 and EMDB-3 are potent inhibitors of enzymes responsible for endomorphin cleavage. These compounds are stable and easily synthesized. EMDB-2 and EMDB-3 are competitive inhibitors of both, DPP IV and APM, with K(i) values in micromolar range. They are less potent than diprotin A in protecting EMs against DPP IV but more potent than actinonin in protecting these peptides against APM.

Natural and synthetic α-methylenelactones and α-methylenelactams with anticancer potential.

α-Methylene-γ- and δ-lactones, as well as α-methylene-γ- and δ-lactams, are plant-derived compounds often used in traditional medicine for the treatment of inflammatory diseases. In recent years, the anticancer properties of these compounds and the molecular mechanisms of their action have been studied extensively. In the search for modern anticancer drugs, various synthetic analogs of α-methylene-γ- and δ-lactones and lactams have been synthesized and tested for their cytotoxic activity. In this review, we give a brief description of the occurrence and biological activity of such compounds isolated from plants and their diverse synthetic analogs.

Comparison of anti-invasive activity of parthenolide and 3-isopropyl-2-methyl-4-methyleneisoxazolidin-5-one (MZ-6)--a new compound with α-methylene-γ-lactone motif--on two breast cancer cell lines.

The biological activities of parthenolide, a sesquiterpene lactone isolated from feverfew, have been attributed to the presence of the α-methylene-γ-lactone skeleton. The lactone skeleton can react via the Michael type addition with sulfhydryl groups of enzymes and other functional proteins, interfering with key biological processes in the cell. In the present study, we describe an efficient method of preparation of 3-isopropyl-2-methyl-4-methyleneisoxazolidin-5-one (MZ-6), a synthetic compound with α-methylene-γ-lactone ring, as in parthenolide, additionally modified by introduction of a nitrogen atom. Furthermore, we investigated the cytotoxic activity and anti-metastatic potential of MZ-6 in comparison with parthenolide. Both compounds showed considerable cytotoxicity against breast cancer MCF-7 and MDA-MB-231 adenocarcinoma cells in vitro and were then evaluated for their anti-metastatic potential. The experimental results showed that MZ-6 and parthenolide suppressed, to a similar degree, migration of MCF-7, but not more aggressive MDA-MB-231 cells. In both cell lines, tested compounds down-regulated mRNA and protein levels of metalloproteinase-9 and urokinase plasminogen activator, the key proteases involved in the degradation of extracellular matrix and dissemination of cancer cells. The obtained results indicate that simple analogs of α-methylene-γ-lactones can be good substitutes for more complex structures isolated from plants.

Effect of 2',6'-dimethyl-L-tyrosine (Dmt) on pharmacological activity of cyclic endomorphin-2 and morphiceptin analogs.

This study reports the synthesis and biological evaluation of a series of new side-chain-to-side-chain cyclized endomorphin-2 (EM-2) and morphiceptin analogs of a general structure Tyr-c(Xaa-Phe-Phe-Yaa)NH(2) or Tyr-c(Xaa-Phe-D-Pro-Yaa)NH(2), respectively, where Xaa and Yaa were L/D Asp or L/D Lys. Further modification of these analogs was achieved by introduction of 2',6'-dimethyl-L-tyrosine (Dmt) instead of Tyr in position 1. Peptides were synthesized by solid phase method and cleaved from the resin by a microwave-assisted procedure. Dmt(1)-substituted analogs displayed high affinity at the µ-opioid receptors, remained intact after incubation with the rat brain homogenate and showed remarkable, long-lasting µ-opioid receptor-mediated antinociceptive activity after central, but not peripheral administration. Our results demonstrate that cyclization is a promising strategy in the development of new opioid analgesics, but further modifications are necessary to enhance the blood-brain barrier permeability.