Synthesis,in vitro stability,and antiproliferative effect of d‐cysteine modified GnRH‐doxorubicin conjugates

Overexpression of gonadotropin‐releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH, [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH, and [d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH were conjugated with doxorubicin (Dox), respectively, through N‐succinimidyl‐3‐maleimidopropionate as a linker to afford three new GnRH‐Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP‐HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor‐positive MCF‐7 human breast cancer cell line by MTT assay. The three GnRH‐Dox conjugates showed improved metabolic stability in human serum in comparison with AN‐152. The antiproliferative effect of conjugate II ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH‐Dox) on MCF‐7 cells was higher than that of conjugate I ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH‐Dox) and conjugate III ([d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH‐Dox), and the cytotoxicity of conjugate II against GnRH receptor‐negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.     

Binding thermodynamics of paromomycin,neomycin, neomycin‐dinucleotide and ‐diPNA conjugates to bacterial and human rRNA

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target‐drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A‐site, with two well‐known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin‐dinucleotide and ‐diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin‐diPNA conjugates as the best candidates for antimicrobial activity. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification and stabilization of a highly selective gastrin‐releasing peptide receptor agonist

The gastrin‐releasing peptide receptor (GRPR) is part of the bombesin receptor family and a well‐known target in cancer diagnosis and therapy. In the last decade, promising results have been achieved by using peptide‐drug conjugates, which allow selective targeting of GRPR expressing tumor cells. Most ligands, however, have been antagonists even though agonists can lead to higher tumor uptake owing to their internalization. So far, only a few studies focused on the identification of small GRPR‐selective agonists that are metabolically stable. Here, we developed novel bombesin analogs with high selectivity for the GRPR and improved blood plasma stability. The most promising analog [d ‐Phe⁶, β‐Ala¹¹, NMe‐Ala¹³, Nle¹⁴]Bn(6‐14) displays an activity of 0.3nM at the GRPR, a more than 4000‐fold selectivity over the other two bombesin receptors and more than 75% stability in human blood plasma after 24 hours. This analog is proposed as a promising drug shuttle for the intracellular delivery of different payloads in targeted tumor therapy approaches.     

Structural requirements for cellular uptake of α‐helical amphipathic peptides

The structure of the cell‐permeable α‐helical amphipathic model peptide FLUOS‐KLALKLALKALKAALKLA‐NH₂ ( I ) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 μm . These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non‐amphipathic 18‐mer peptides with different primary structure, net charge and helix parameters from I . The amphipathic counterparts were internalized into the cells to a comparable extent as I , whereas no cellular uptake could be detected for the non‐amphipathic analogues. The mode of uptake remains unclear and involves both temperature‐sensitive and ‐insensitive processes, indicating non‐endocytic contributions. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Z‐FA.FMK activates duodenal epithelial cell proliferation through oxidative stress,NF‐κB and IL‐1β in d‐GalN/TNF‐α‐administered mice

This study was designed to evaluate the effect of Z‐FA.FMK (benzyloxycarbonyl‐l ‐phenylalanyl‐alanine‐fluoromethylketone), a pharmacological inhibitor of cathepsin B, on the proliferation of duodenal mucosal epithelial cells and the cellular system that controls this mechanism in these cells in vivo. For this investigation, BALB/c male mice were divided into four groups. The first group received physiological saline, the second group was administered Z‐FA.FMK, the third group received d ‐GalN (d ‐galactosamine) and TNF‐α (tumour necrosis factor‐α) and the fourth group was given both d ‐GalN/TNF‐α and Z‐FA.FMK. When d ‐GalN/TNF‐α was administered alone, we observed an increase in IL‐1β‐positive and active NF‐κB‐positive duodenal epithelial cells, a decrease in PCNA (proliferative cell nuclear antigen)‐positive duodenal epithelial cells and an increase in degenerative changes in duodenum. On the other hand, Z‐FA.FMK pretreatment inhibited all of these changes. Furthermore, lipid peroxidation, protein carbonyl and collagen levels were increased, glutathione level and superoxide dismutase activity were decreased, while there was no change in catalase activity by d ‐GalN/TNF‐α injection. On the contrary, the Z‐FA.FMK pretreatment before d ‐GalN/TNF‐α blocked these effects. Based on these findings, we suggest that Z‐FA.FMK might act as a proliferative mediator which is controlled by IL‐1β through NF‐κB and oxidative stress in duodenal epithelial cells of d ‐GalN/TNF‐α‐administered mice.     

Methoxinine – an alternative stable amino acid substitute for oxidation‐sensitive methionine in radiolabelled peptide conjugates

Radiolabelled peptides with high specificity and affinity towards receptors that are overexpressed by tumour cells are used in nuclear medicine for the diagnosis (imaging) and therapy of cancer. In some cases, the sequences of peptides under investigations contain methionine (Met), an amino acid prone to oxidation during radiolabelling procedures. The formation of oxidative side products can affect the purity of the final radiopharmaceutical product and/or impair its specificity and affinity towards the corresponding receptor. The replacement of Met with oxidation resistant amino acid analogues, for example, norleucine (Nle), can provide a solution. While this approach has been applied successfully to different radiolabelled peptides, a Met → Nle switch only preserves the length of the amino acid side chain important for hydrophobic interactions but not its hydrogen‐bonding properties. We report here the use of methoxinine (Mox), a non‐canonical amino acid that resembles more closely the electronic properties of Met in comparison to Nle. Specifically, we replaced Met¹⁵ by Mox¹⁵ and Nle¹⁵ in the binding sequence of a radiometal‐labelled human gastrin derivative [d ‐Glu¹⁰]HG(10‐17), named MG11 (d ‐Glu‐Ala‐Tyr‐Gly‐Trp‐Met‐Asp‐Phe‐NH₂). A comparison of the physicochemical properties of ¹⁷⁷Lu‐DOTA[ X ¹⁵]MG11 ( X = Met, Nle, Mox) in vitro (cell internalization/externalization properties, receptor affinity (IC₅₀), blood plasma stability and logD) showed that Mox indeed represents a suitable, oxidation‐stable amino acid substitute of Met in radiolabelled peptide conjugates. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

The glucose‐deprivation network counteracts lapatinib‐induced toxicity in resistant ErbB2‐positive breast cancer cells

Dynamic interactions between intracellular networks regulate cellular homeostasis and responses to perturbations. Targeted therapy is aimed at perturbing oncogene addiction pathways in cancer, however, development of acquired resistance to these drugs is a significant clinical problem. A network‐based computational analysis of global gene expression data from matched sensitive and acquired drug‐resistant cells to lapatinib, an EGFR/ErbB2 inhibitor, revealed an increased expression of the glucose deprivation response network, including glucagon signaling, glucose uptake, gluconeogenesis and unfolded protein response in the resistant cells. Importantly, the glucose deprivation response markers correlated significantly with high clinical relapse rates in ErbB2‐positive breast cancer patients. Further, forcing drug‐sensitive cells into glucose deprivation rendered them more resistant to lapatinib. Using a chemical genomics bioinformatics mining of the CMAP database, we identified drugs that specifically target the glucose deprivation response networks to overcome the resistant phenotype and reduced survival of resistant cells. This study implicates the chronic activation of cellular compensatory networks in response to targeted therapy and suggests novel combinations targeting signaling and metabolic networks in tumors with acquired resistance.     

Dynamin‐related protein 1‐mediated mitochondrial fission contributes to IR‐783‐induced apoptosis in human breast cancer cells

IR‐783 is a kind of heptamethine cyanine dye that exhibits imaging, cancer targeting and anticancer properties. A previous study reported that its imaging and targeting properties were related to mitochondria. However, the molecular mechanism behind the anticancer activity of IR‐783 has not been well demonstrated. In this study, we showed that IR‐783 inhibits cell viability and induces mitochondrial apoptosis in human breast cancer cells. Exposure of MDA‐MB‐231 cells to IR‐783 resulted in the loss of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) depletion, mitochondrial permeability transition pore (mPTP) opening and cytochrome c (Cyto C) release. Furthermore, we found that IR‐783 induced dynamin‐related protein 1 (Drp1) translocation from the cytosol to the mitochondria, increased the expression of mitochondrial fission proteins mitochondrial fission factor (MFF) and fission‐1 (Fis1), and decreased the expression of mitochondrial fusion proteins mitofusin1 (Mfn1) and optic atrophy 1 (OPA1). Moreover, knockdown of Drp1 markedly blocked IR‐783‐mediated mitochondrial fission, loss of MMP, ATP depletion, mPTP opening and apoptosis. Our in vivo study confirmed that IR‐783 markedly inhibited tumour growth and induced apoptosis in an MDA‐MB‐231 xenograft model in association with the mitochondrial translocation of Drp1. Taken together, these findings suggest that IR‐783 induces apoptosis in human breast cancer cells by increasing Drp1‐mediated mitochondrial fission. Our study uncovered the molecular mechanism of the anti‐breast cancer effects of IR‐783 and provided novel perspectives for the application of IR‐783 in the treatment of breast cancer.     

Transforming growth factor‐β1 modulates metalloproteinase‐2 and ‐9, nitric oxide,RhoA and α‐smooth muscle actin expression in colon adenocarcinoma cells

Colon carcinoma invasiveness is a process involving cell–cell and cell–matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF‐β1 (transforming growth factor‐β1) and small G protein RhoA in tumour progression, the influence of TGF‐β1 treatment or RhoA‐associated kinase inhibitor on the production of NO (nitric oxide) and MMP‐2 and MMP‐9 (metalloproteinases‐2 and ‐9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP‐2 and MMP‐9. rhTGF‐β1 and the synthetic Rho kinase inhibitor (Y‐27632) decreased MMP‐2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF‐β1 decreased NO secretion by cells, while Y‐27632 had no effect on it. Immunoblotting with anti‐RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF‐β1. rhTGF‐β1 induced α‐smooth muscle actin (α‐SMA) expression, especially in high Duke's grade of colon cancer, while Y‐27632 blocked it. Summing up, in colon carcinoma cells, TGF‐β1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and α‐SMA expression or assayed using motility data and may be a good target for cancer therapy.     

A stable meta‐carborane enables the generation of boron‐rich peptide agonists targeting the ghrelin receptor

Boron neutron capture therapy (BNCT) is a binary cancer therapy, which combines the biochemical targeting of a boron‐containing drug with the regional localization of radiation treatment. Although the concept of BNCT has been known for decades, the selective delivery of boron into tumor cells remains challenging. G protein‐coupled receptors that are overexpressed on cancer cells in combination with peptidic ligands can be potentially used as shuttle system for a tumor‐directed boron uptake. In this study, we present the generation of short, boron‐rich peptide conjugates that target the ghrelin receptor. Expression of the ghrelin receptor on various cancer cells makes it a viable target for BNCT. We designed a novel hexapeptide super‐agonist that was modified with different specifically synthesized carborane monoclusters and tested for ghrelin receptor activation. A meta‐carborane building block with a mercaptoacetic acid linker was found to be optimal for peptide modification, owing to its chemical stability and a suitable activation efficacy of the conjugate. The versatility of this carborane for the development of peptidic boron delivery agents was further demonstrated by the generation of highly potent, boron‐loaded conjugates using the backbone of the known ghrelin receptor ligands growth hormone releasing peptide 6 and Ipamorelin.     

Conformational transformation of ascidiacyclamide analogues induced by incorporating enantiomers of phenylalanine, 1‐naphthylalanine or 2‐naphthylalanine

We designed five ascidiacyclamide analogues [cyclo(‐Xxx¹‐oxazoline²‐d ‐Val³‐thiazole⁴‐l ‐Ile⁵‐oxazoline⁶‐d ‐Val⁷‐thiazole⁸‐)] incorporating l ‐1‐naphthylalanine (l ‐1Nal), l ‐2‐naphthylalanine (l ‐2Nal), d ‐phenylalanine (d ‐Phe), d ‐1‐naphthylalanine (d ‐1Nal) or d ‐2‐naphthylalanine (d ‐2Nal) into the Xxx¹ position of the peptide. The conformations of these analogues were then examined using ¹H NMR, CD spectroscopy, and X‐ray diffraction. These analyses suggested that d ‐enantiomer‐incorporated ASCs [(d ‐Phe), (d ‐1Nal), and (d ‐2Nal)ASC] transformed from the folded to the open structure in solution more easily than l ‐enantiomer‐incorporated ASCs [(l ‐Phe), (l ‐1Nal), and (l ‐2Nal)ASC]. Structural comparison of the two analogues containing isomeric naphthyl groups showed that the 1‐naphthyl isomer induced a more stable open structure than the 2‐naphthyl isomer. In particular, [d ‐1Nal]ASC showed the most significant transformation from the folded to the open structure in solution, and exhibited the strongest cytotoxicity toward HL‐60 cells. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Cellular uptake and biological activity of peptide nucleic acids conjugated with peptides with and without cell‐penetrating ability

A 12‐mer peptide nucleic acid (PNA) directed against the nociceptin/orphanin FQ receptor mRNA was disulfide bridged with various peptides without and with cell‐penetrating features. The cellular uptake and the antisense activity of these conjugates were assessed in parallel. Quantitation of the internalized PNA was performed by using an approach based on capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF). This approach enabled a selective assessment of the PNA moiety liberated from the conjugate in the reducing intracellular environment, thus avoiding bias of the results by surface adsorption. The biological activity of the conjugates was studied by an assay based on the downregulation of the nociceptin/orphanin FQ receptor in neonatal rat cardiomyocytes (CM). Comparable cellular uptake was found for all conjugates and for the naked PNA, irrespective of the cell‐penetrating properties of the peptide components. All conjugates exhibited a comparable biological activity in the 100 nM range. The naked PNA also exhibited extensive antisense activity, which, however, proved about five times lower than that of the conjugates. The found results suggest cellular uptake and the bioactivity of PNA‐peptide conjugates to be not primarily related to the cell‐penetrating ability of their peptide components. Likewise from these results it can be inferred that the superior bioactivity of the PNA‐peptide conjugates in comparison with that of naked PNA rely on as yet unknown factors rather than on higher membrane permeability. Several hints point to the resistance against cellular export and the aggregation propensity combined with the endocytosis rate to be candidates for such factors. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

TLR3 engagement induces IRF‐3‐dependent apoptosis in androgen‐sensitive prostate cancer cells and inhibits tumour growth in vivo

Toll‐like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double‐stranded RNA analogue poly I:C induces apoptosis of androgen‐sensitive prostate cancer (PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3‐mediated apoptosis and the in vivo efficacy of poly I:C‐based therapy. We show that interferon regulatory factor‐3 (IRF‐3) signalling plays an essential role in TLR3‐mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE‐1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well‐established human androgen‐sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C‐treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF‐3 in both human normal and PCa clinical samples, potentially envisaging poly I:C‐based therapy for PCa.     

Interactions of vasopressin and oxytocin receptors with vasopressin analogues substituted in position 2 with 3,3′‐diphenylalanine – a molecular docking study

Vasopressin and oxytocin receptors belong to the superfamily of G protein‐coupled receptors and play an important role in many physiological functions. They are also involved in a number of pathological conditions being important drug targets. In this work, four vasopressin analogues substituted at position 2 with 3,3′‐diphenylalanine have been docked into partially flexible vasopressin and oxytocin receptors. The bulky residue at position 2 acts as a structural restraint much stronger in the oxytocin receptor (OTR) than in the vasopressin V2 receptor (V2R), resulting in a different location of the analogues in these receptors. This explains the different, either agonistic or antagonistic, activities of the analogues in V2R and OTR, respectively. In all complexes, the conserved polar residues serve as anchor points for the ligand both in OTR and V2R. Strong interactions of the C‐terminus of analogue II ([Mpa¹,d ‐Dpa²,Val⁴,d ‐Arg⁸]VP) with extracellular loop 3 may be responsible for its highest activity at V2R. It also appears that V2R adapts more readily to the docking analogues by conformational changes in the aromatic side chains triggering receptor activation. A weak activity at V1a vasopressin receptor appears to be caused by weak receptor–ligand interactions. Results of this study may facilitate a rational design of new analogues with the highest activity/selectivity at vasopressin and OTRs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Design, synthesis, and in vitro activity of novel drug delivery systems containing tuftsin derivatives and methotrexate

During the past decade, biodegradable polymers or oligopeptides recognized by cell-surface receptors have been shown to increase drug specificity, lowering systemic drug toxicity in contrast to small-size fast-acting drugs. The goal of the present study was to develop anticancer bioconjugates based on chemotactic drug targeting (CDT). These constructs are composed of methotrexate (Mtx) attached to a tuftsin-like peptide carrier through an enzyme-labile pentapeptide spacer (GFLGC) and several copies of a chemotactic targeting moiety (H-TKPR, For-TKPR, H-TKPKG, and Ac-TKPKG). Carriers with targeting moieties in the branches were prepared by solid-phase synthesis using mixed Boc and Fmoc strategies. The drug molecule connected to an enzyme-labile spacer was attached to the branched oligopeptide in solution. In vitro chemotaxis, cellular uptake, and cytotoxicity assays were carried out on the MonoMac6 cell line. The most effective conjugates with H-TKPR or Ac-TKPKG targeting moieties in the branches, which have the most advantageous chemotactic properties, can be internalized rapidly, and these conjugates trigger higher toxic effect than the free drug (Mtx). The results suggest that our tuftsin-based drug delivery systems might be potential candidates for targeting cancer chemotherapy.     

TFPI‐2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour‐stromal cell interactions

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI‐2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI‐2 is frequently down‐regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI‐2 down‐regulation in the National Cancer Institute (NCI)‐H460 non‐small cell lung cancer cell line using specific micro interfering micro‐interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI‐2 down‐regulation enhanced cell adhesion to collagen IV and laminin via an increase in α₁ integrin on cell surface, and increased MMP expression (mainly MMP‐1 and ‐3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI‐2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP‐1, ‐3 and ‐7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.     

A molecular understanding of d‐homoestrone‐induced G2/M cell cycle arrest in HeLa human cervical carcinoma cells

2‐Methoxyestradiol (ME), one of the most widely investigated A‐ring‐modified metabolites of estrone, exerts significant anticancer activity on numerous cancer cell lines. Its pharmacological actions, including cell cycle arrest, microtubule disruption and pro‐apoptotic activity, have already been described in detail. The currently tested d ‐ring‐modified analogue of estrone, d ‐homoestrone, selectively inhibits cervical cancer cell proliferation and induces a G2/M phase cell cycle blockade, resulting in the development of apoptosis. The question arose of whether the difference in the chemical structures of these analogues can influence the mechanism of anticancer action. The aim of the present study was therefore to elucidate the molecular contributors of intracellular processes induced by d ‐homoestrone in HeLa cells. Apoptosis triggered by d ‐homoestrone develops through activation of the intrinsic pathway, as demonstrated by determination of the activities of caspase‐8 and ‐9. It was revealed that d ‐homoestrone‐treated HeLa cells are not able to enter mitosis because the cyclin‐dependent kinase 1‐cyclin B complex loses its activity, resulting in the decreased inactivation of stathmin and a concomitant disturbance of microtubule formation. However, unlike 2‐ME, d ‐homoestrone does not exert a direct effect on tubulin polymerization. These results led to the conclusion that the d ‐homoestrone‐triggered intracellular processes resulting in a cell cycle arrest and apoptosis in HeLa cells differ from those in the case of 2‐ME. This may be regarded as an alternative mechanism of action among steroidal anticancer compounds.     

Investigation on chemotactic drug targeting (chemotaxis and adhesion) inducer effect of GnRH‐III derivatives in Tetrahymena and human leukemia cell line

GnRH‐III has been shown to exert a cytotoxic effect on the GnRH‐R positive tumor cells. The chemotactic drug targeting (CDT) represents a new way for drug delivery approach based on selective chemoattractant guided targeting. The major goal of the present work was to develop and investigate various GnRH‐III derivatives as potential targeting moieties for CDT. The cell physiological effects (chemotaxis, adhesion, and signaling) induced by three native GnRHs (hGnRH‐I, cGnRH‐II, and lGnRH‐III) and nine GnRH‐III derivatives were evaluated in two model cells (Tetrahymena pyriformis and Mono Mac 6 human monocytes). According to our results, the native GnRH‐III elicited the highest chemoattractant and adhesion inducer activities of all synthesized peptides in micromolar concentrations in monocytes. With respect to chemoattraction, dimeric derivatives linked by a disulfide bridge ([GnRH‐III(C)]₂) proved to be efficient in both model cells; furthermore, acetylation of the linker region ([GnRH‐III(Ac‐C)]₂) could slightly improve the chemotactic and adhesion effects in monocytes. The length of the peptide and the type of N‐terminal amino acid could also determine the chemotactic and adhesion modulation potency of each fragment. The application of the chemoattractant GnRH‐III derivatives was accompanied by a significant activation of phosphatidylinositol 3‐kinase in both model cells. In summary, our work on low‐level differentiated model cells of tumors has proved that GnRH‐III and some of its synthetic derivatives are promising candidates to be applied in CDT: these compounds might act both as carrier, delivery unit, and antitumor agents. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

MARCH1 encourages tumour progression of hepatocellular carcinoma via regulation of PI3K‐AKT‐β‐catenin pathways

Membrane‐associated RING‐CH‐1 (MARCH1) is a membrane‐anchored E3 ubiquitin ligase that is involved in a variety of cellular processes. MARCH1 was aberrantly expressed as a tumour promoter in ovarian cancer, but the signalling about the molecular mechanism has not yet been fully illuminated. Here, we first determined that MARCH1 was obviously highly expressed in human hepatocellular carcinoma samples and cells. In addition, our findings demonstrated that the proliferation, migration and invasion of hepatocellular carcinoma were suppressed, but the apoptosis was increased, as a result of MARCH1 knockdown by either siRNA targeting MARCH1 or pirarubicin treatment. Conversely, the proliferation, migration and invasion of hepatocellular carcinoma were obviously accelerated, and the apoptosis was decreased, by transfecting the MARCH1 plasmid to make MARCH1 overexpressed. Moreover, in vivo, the results exhibited a significant inhibition of the growth of hepatocellular carcinoma in nude mice, which were given an intra‐tumour injection of siRNA targeting MARCH1. Furthermore, our study concluded that MARCH1 functions as a tumour promoter, and its role was up‐regulated the PI3K‐AKT‐β‐catenin pathways both in vitro and in vivo. In summary, our work determined that MARCH1 has an important role in the development and progression of hepatocellular carcinoma and may be used as a novel potential molecular therapeutic target in the future treatment of hepatocellular carcinoma.