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 biological evaluation of α-methylidene-δ-lactones with 3,4-dihydrocoumarin skeleton.

A series of new 3-methylidenechroman-2-ones bearing various aromatic moieties and various substituents at position 4 were synthesized in a three step reaction sequence. Friedel-Crafts alkylation of phenols or naphthols using ethyl 3-methoxy-2-diethoxyphosphorylacrylate in the presence of trifluoromethanesulphonic acid gave 3-diethoxyphosphorylchromen-2-ones. These compounds were employed as Michael acceptors in the reaction with Grignard reagents to give adducts which were finally used as Horner-Wadsworth-Emmons reagents for the olefination of formaldehyde. All obtained 3-methylidenechroman-2-ones were tested against two human leukemia cell lines NALM-6 and HL-60 as well as MCF-7 breast cancer and HT-29 colon cancer adenocarcinomas. Several obtained methylidenechromanones displayed high cytotoxic activity with IC(50) values below 1 µM, mainly against leukemia and MCF-7 cell lines. Investigation of structure-activity relationships revealed that the presence of additional, ortho-fused benzene ring and n-butyl or i-propyl group in position 4 enhances the activity. Selected methylidenechromanones were also tested on normal human umbilical vein endothelial cells (HUVEC) and chromanone 14o was found to be eightfold more toxic against MCF-7 than normal cells. Furthermore, antimicrobial assays revealed that chromanone 14n is highly active and bactericidal at concentration equal to MIC or 2MIC against nosocomial and community-associated staphylococci (MRSA) which are resistant to most or all available therapeutic classes of antimicrobial drugs.

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.

The influence of opioids on matrix metalloproteinase-2 and -9 secretion and mRNA levels in MCF-7 breast cancer cell line.

Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in degradation of extracellular matrix, a process that initiates uncontrolled spread of proliferating cancer cells and therefore plays a crucial role in cancer invasion and metastasis. Compounds able to modulate MMP activity may become important tools in cancer research. In the present study we examined the effect of two µ-selective opioids, morphine and endomorphin-2 (EM-2) on the production of MMP-2 and MMP-9 in MCF-7 cells. We report that both opioids time- and concentration-dependently inhibited the expression and secretion of these MMPs. The observed effect was not reversed by naloxone (Nal). Further experiments showed that morphine and EM-2 decreased endothelial nitric oxide synthase (eNOS) mRNA level and nitric oxide (NO) secretion in MCF-7 cells. These findings indicate that attenuation of MMP secretion by opioids was not mediated by opioid receptors but was under the control of nitric oxide system.

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.

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.

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.

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.

The role of morphine in regulation of cancer cell growth.

Morphine is considered the "gold standard" for relieving pain and is currently one of the most effective drugs available clinically for the management of severe pain associated with cancer. In addition to its use in the treatment of pain, morphine appears to be important in the regulation of neoplastic tissue. Although morphine acts directly on the central nervous system to relieve pain, its activities on peripheral tissues are responsible for many of the secondary complications. Therefore, understanding the impact, other than pain control, of morphine on cancer treatment is extremely important. The effect of morphine on tumor growth is still contradictory, as both growth-promoting and growth-inhibiting effects have been observed. Accumulating evidence suggests that morphine can affect proliferation and migration of tumor cells as well as angiogenesis. Various signaling pathways have been suggested to be involved in these extra-analgesic effects of morphine. Suppression of immune system by morphine is an additional complication. This review provides an update on the influence of morphine on the growth and migration potential of tumor cells.

Effect of potent endomorphin degradation blockers on analgesic and antidepressant-like responses in mice.

The biological effects of endomorphins (EMs) are short-lasting due to their rapid degradation by endogenous enzymes. Competing enzymatic degradation is an approach to prolong EM bioavailability. In the present study, a series of tetra- and tripeptides of similar to EMs structure was synthesized and tested in vitro and in vivo for their ability to inhibit degradation of EMs. The obtained results indicated that, among the series of analogs, the tetrapeptide Tyr-Pro-d-ClPhe-Phe-NH(2) and the tripeptide Tyr-Pro-Ala-NH(2), which did not bind to the µ-opioid receptors, were potent inhibitors of EM catabolism in rat brain homogenate. In vivo, these two peptides significantly prolonged the analgesic and antidepressant-like effects, induced by exogenous EMs, by blocking EM degrading enzymes. These new potent inhibitors may therefore increase the level and the half life of endogenous EMs and could be used in a new therapeutic strategy against pain and mood disorders, based on increasing of EM bioavailability.

Synthesis and biological evaluation of cyclic endomorphin-2 analogs.

In our previous paper we reported synthesis and biological activity of two cyclic analogs of endomorphin-2 (EM-2): Tyr-c(Lys-Phe-Phe-Asp)-NH(2) and Tyr-c(Asp-Phe-Phe-Lys)-NH(2), achieved by making an amid bond between Lys and Asp side-chains. The first analog did not bind to the mu-opioid receptor, the affinity of the second one was very low. In the present study, we describe the synthesis of four novel cyclic analogs of similar structure, but with d-amino acids in position 2 (D-Lys or D-Asp). All new analogs displayed high affinity for the mu-opioid receptor, were much more stable than EM-2 in rat brain homogenate and showed remarkable antinociceptive activity after intracerebroventricular (i.c.v.) administration. Analgesic effect of the most potent cyclic analog, Tyr-c(D-Lys-Phe-Phe-Asp)NH(2) was much stronger and longer lasting than that of EM-2. This analog elicited analgesia also after peripheral administration and this effect was reversed by concomitant i.c.v. injection of the mu-opioid antagonist, beta-funaltrexamine, which indicated that antinociception was mediated by the mu-opioid receptor in the brain. Central action of the cyclic analog gives evidence that it was able to cross the blood-brain barrier, most likely due to the increased lipophilicity. Our results demonstrate that cyclization might be a promising strategy to enhance bioavailability of peptides and may serve a role in the development of novel endomorphin analogs with increased therapeutic potential.

Design, synthesis and pharmacological characterization of endomorphin analogues with non-cyclic amino acid residues in position 2.

A series of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) analogues, containing non-cyclic amino acids (Ala, D-Ala, beta-Ala, NMeAla, D-NMeAla or Sar) instead of Pro in position 2 was synthesized, where NMeAla = N-methylalanine and Sar = N-methylglycine, sarcosine. The opioid activity profiles of these peptides were determined in mu and delta opioid receptor (MOR and DOR)-representative binding assays and bioassays in vitro, as well as in the mouse hot-plate test in vivo. Finally, the degradation rates of all analogues in the presence of either rat brain homogenate or selected proteolytic enzymes were determined. Analogues of EM-2 were generally more potent than the respective analogues of EM-1. EM-2 analogues with D-Ala or D-NMeAla were about twofold more potent than the parent peptide and were least prone to degradation by brain homogenate, dipeptydyl peptidase IV and aminopeptidase M. In the in vivo test, [D-Ala(2)]EM-2 and [D-NMeAla(2)]EM-2 showed much higher analgesic potency than EM-2 which confirmed the usefulness of structural modifications in obtaining new leads for pain-relief therapeutics.