The epichaperome is an integrated chaperome network that facilitates tumour survival.

Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes-dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically 'rewired' to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.

Molecular mode of action of NKP-1339 - a clinically investigated ruthenium-based drug - involves ER- and ROS-related effects in colon carcinoma cell lines.

Sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (NKP-1339) is a clinically investigated ruthenium-based metal complex, which shows promising results in solid tumors, such as non-small cell lung cancer, colorectal carcinoma, and most distinctively in gastrointestinal neuroendocrine tumors. In previous studies, fast binding to albumin as well as transferrin could be shown. The enhanced permeability and retention (EPR) effect, which is diversely being exploited for tumor targeting, could therefore be applicable for NKP-1339. Here we studied the serum dependence of its biological activity in various methods, influencing its cellular accumulation, cytotoxicity as well as the generation of reactive oxygen species (ROS). ROS lead to Nrf2 activation, which is known to activate antioxidant response gene transcription. GRP78 down-regulation on the protein level suggests ER associated protein degradation (ERAD) as a mode of action, as RNA levels are only mildly affected. Another important part for the mode of action is endoplasmic reticulum (ER) stress, as different factors are highly upregulated on the protein level. For example PERK, a transmembrane receptor which is released by GRP78 when the ER is disturbed, is upregulated and phosphorylated. EIF2α is phosphorylated, which leads to an inhibition of CAP-dependent translation and other stress responses. The transcription factor CHOP (DDIT3), which promotes ER stress dependent apoptosis, is time and concentration dependently upregulated. Finally cytotoxicity tests could prove that inhibition of ER stress and ER stress-mediated apoptosis leads to decreased cytotoxic effects of NKP-1339, which highlights the involvement of this mechanism in the mode of action.

Three-dimensional and co-culture models for preclinical evaluation of metal-based anticancer drugs.

BACKGROUND: Hypoxic and necrotic regions that accrue within solid tumors in vivo are known to be associated with metastasis formation, radio- and chemotherapy resistance, and drug metabolism. Therefore, integration of these tumor characteristics into in vitro drug screening models is advantageous for any reliable investigation of the anticancer activity of novel drug candidates. In general, usage of cell culture models with in vivo like characteristics has become essential in preclinical drug studies and allows evaluation of complex problems such as tumor selectivity and anti-invasive properties of the drug candidates. MATERIALS AND METHODS: In this study, we investigated the anticancer activity of clinically approved, investigational and experimental drugs based on platinum (cisplatin, oxaliplatin and KP1537), gallium (KP46), ruthenium (KP1339) and lanthanum (KP772) in different cell culture models such as monolayers, multicellular spheroids, as well as invasion and metastasis models. Results Application of the Alamar Blue assay to multicellular spheroids and a spheroid-based invasion assay resulted in an altered rating of compounds with regard to their cytotoxicity and ability to inhibit invasion when compared with monolayer-based cytotoxicity and transwell assays. For example, the gallium-based drug candidate KP46 showed in spheroid cultures significantly enhanced properties to inhibit protrusion formation and fibroblast mediated invasiveness, and improved cancer cell selectivity. CONCLUSION: Taken together, our results demonstrate the advantages of spheroid-based assays and underline the necessity of using different experimental models for reliable preclinical investigations assessing and better predicting the anticancer potential of new compounds.

Triapine and a more potent dimethyl derivative induce endoplasmic reticulum stress in cancer cells.

Triapine (3-AP; 3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a ribonucleotide reductase inhibitor, has been extensively evaluated in clinical trials in the last decade. This study addresses the role of endoplasmic reticulum (ER) stress in the anticancer activity of 3-AP and the derivative N(4),N(4)-dimethyl-triapine (3-AP-Me), differing from 3-AP only by dimethylation of the terminal nitrogen. Treatment of colon cancer cells with 3-AP or 3-AP-Me activated all three ER stress pathways (PERK, IRE1a, ATF6) by phosphorylation of eIF2α and upregulation of gene expression of activating transcription factors ATF4 and ATF6. In particular, 3-AP-Me led to an upregulation of the alternatively spliced mRNA variant XBP1 (16-fold). Moreover, 3-AP and 3-AP-Me activated the cellular stress kinases c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases, and inhibition of JNK activity antagonized the cytotoxic effect of both compounds. Subsequent to induction of the unfolded protein response, a significant upregulation of proapoptotic proteins was detected, including the transcription factor CHOP and Bim, an essential factor for ER stress-related apoptosis. In correlation with the higher degree of ER stress after 3-AP-Me treatment, also a more potent depolarization of mitochondrial membranes was found. These data suggest that 3-AP and 3-AP-Me induce apoptosis via ER stress. This was further corroborated by showing that inhibition of protein biosynthesis with cycloheximide prior to 3-AP and 3-AP-Me treatment leads to a significant reduction of the antiproliferative properties of both compounds. Taken together, this study demonstrates that induction of ER stress contributes to the mode of action of 3-AP and that terminal dimethylation leads to an even more pronounced manifestation of this effect.

Copper(II) complexes with highly water-soluble L- and D-proline-thiosemicarbazone conjugates as potential inhibitors of Topoisomerase IIα.

Two proline-thiosemicarbazone bioconjugates with excellent aqueous solubility, namely, 3-methyl-(S)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone [L-Pro-FTSC or (S)-H2L] and 3-methyl-(R)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone [D-Pro-FTSC or (R)-H2L], have been synthesized and characterized by elemental analysis, one- and two-dimensional (1)H and (13)C NMR spectroscopy, and electrospray ionization mass spectrometry. The complexation behavior of L-Pro-FTSC with copper(II) in an aqueous solution and in a 30% (w/w) dimethyl sulfoxide/water mixture has been studied via pH potentiometry, UV-vis spectrophotometry, electron paramagnetic resonance, (1)H NMR spectroscopy, and spectrofluorimetry. By the reaction of copper(II) acetate with (S)-H2L and (R)-H2L in water, the complexes [Cu(S,R)-L] and [Cu(R,S)-L] have been synthesized and comprehensively characterized. An X-ray diffraction study of [Cu(S,R)-L] showed the formation of a square-pyramidal complex, with the bioconjugate acting as a pentadentate ligand. Both copper(II) complexes displayed antiproliferative activity in CH1 ovarian carcinoma cells and inhibited Topoisomerase IIα activity in a DNA plasmid relaxation assay.

Mechanisms underlying reductant-induced reactive oxygen species formation by anticancer copper(II) compounds.

Intracellular generation of reactive oxygen species (ROS) via thiol-mediated reduction of copper(II) to copper(I) has been assumed as the major mechanism underlying the anticancer activity of copper(II) complexes. The aim of this study was to compare the anticancer potential of copper(II) complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone; currently in phase II clinical trials) and its terminally dimethylated derivative with that of 2-formylpyridine thiosemicarbazone and that of 2,2'-bipyridyl-6-carbothioamide. Experiments on generation of oxidative stress and the influence of biologically relevant reductants (glutathione, ascorbic acid) on the anticancer activity of the copper complexes revealed that reductant-dependent redox cycling occurred mainly outside the cells, leading to generation and dismutation of superoxide radicals resulting in cytotoxic amounts of H(2)O(2). However, without extracellular reductants only weak intracellular ROS generation was observed at IC(50) levels, suggesting that cellular thiols are not involved in copper-complex-induced oxidative stress. Taken together, thiol-induced intracellular ROS generation might contribute to the anticancer activity of copper thiosemicarbazone complexes but is not the determining factor.

L- and D-proline thiosemicarbazone conjugates: coordination behavior in solution and the effect of copper(II) coordination on their antiproliferative activity.

Two enantiomerically pure thiosemicarbazone-proline conjugates with enhanced aqueous solubility, namely, 2-hydroxy-3-methyl-(S)-pyrrolidine-2-carboxylate-5-methylbenzaldehyde thiosemicarbazone [L-Pro-STSC or (S)-H(2)L] and 2-hydroxy-3-methyl-(R)-pyrrolidine-2-carboxylate-5-methylbenzaldehyde thiosemicarbazone [D-Pro-STSC or (R)-H(2)L] have been synthesized and characterized by elemental analysis, spectroscopic methods (UV-vis and (1)H and (13)C NMR), and electrospray ionization mass spectrometry. The metal complexation behavior of L-Pro-STSC, stoichiometry, and thermodynamic stability of iron(II), iron(III), copper(II), and zinc(II) complexes in 30% (w/w) dimethyl sulfoxide/H(2)O solvent mixture have been studied by pH-potentiometric, UV-vis-spectrophotometric, circular dichroism, electron paramagnetic resonance, (1)H NMR spectroscopic, and spectrofluorimetric measurements. By the reaction of CuCl(2)·2H(2)O with (S)-H(2)L and (R)-H(2)L, respectively, the complexes [Cu[(S)-H(2)L]Cl]Cl and [Cu[(R)-H(2)L]Cl]Cl have been prepared and comprehensively characterized. An X-ray diffraction study of [Cu[(R)-H(2)L]Cl]Cl showed the formation of a square-planar copper(II) complex, which builds up stacks with interplanar separation of 3.3 Å. The antiproliferative activity of two chiral ligands and their corresponding copper(II) complexes has been tested in two human cancer cell lines, namely, SW480 (colon carcinoma) and CH1 (ovarian carcinoma). The thiosemicarbazone-proline conjugates L- and D-Pro-STSC show only moderate cytotoxic potency with IC(50) values of 62 and 75 µM, respectively, in CH1 cells and >100 µM in SW480 cells. However, the corresponding copper(II) complexes are 13 and 5 times more potent in CH1 cells, based on a comparison of IC(50) values, and in SW480 cells the increase in the antiproliferative activity is even higher. In both tested cell lines, L-Pro-STSC as well as its copper(II) complex show slightly stronger antiproliferative activity than the compounds with a D-Pro moiety, yielding IC(50) values of 4.6 and 5.5 µM for [Cu(L-Pro-STSC)Cl]Cl in CH1 and SW480 cells, respectively.

Comparison of nab-paclitaxel plus gemcitabine in elderly versus younger patients with metastatic pancreatic cancer: Analysis of a multicentre, prospective, non-interventional study.

BACKGROUND: Pancreatic cancer (PC) ranks among the deadliest malignancies worldwide. In the MPACT study, first-line nab-paclitaxel plus gemcitabine (nab-P/G) demonstrated activity (median overall survival [OS], 8.7 months) and tolerability in patients with metastatic PC (mPC). However, the clinical evidence of nab-P/G in the elderly (>70 years), who account for the majority of patients with mPC, is limited. This is the first prospective, multicentre, non-interventional study evaluating the tolerability and effectiveness of nab-P/G in younger (≤70 years) versus elderly (>70 years) patients with mPC in the daily clinical routine. METHODS: Eligible patients with mPC were treated with nab-P/G and observed until disease progression or unacceptable toxicity. The primary objectives were safety and tolerability of nab-P/G, and the secondary objectives were efficacy and real-life dosing. RESULTS: A total of 317 patients with mPC (median age, 70 years) were recruited, of which 299, aged ≤70 (n = 162) and >70 (n = 137) years, were eligible for analysis. Baseline characteristics and the safety profile were comparable between the groups. However, fatigue (22.8% versus 13.0%) and decreased appetite (8.8% versus 1.2%) were more frequent in elderly patients. Younger versus elderly patients equally benefited in terms of objective response rate (36% versus 48%), median progression-free survival (5.6 versus 5.5 months; hazard ratio [HR] = 1.03; p = 0.81) and OS (10.6 versus 10.2 months; HR = 0.89; p = 0.4). In addition, the median treatment duration (5 versus 4 cycles), relative dose intensity (70% versus 74%) or reasons for treatment discontinuation were similar. Most patients (56.2% versus 47.4%) benefited from a second-line therapy. CONCLUSION: This prospective real-world analysis confirms the feasibility and tolerability of nab-P/G treatment and reveals OS data similar for younger patients and elderly patients aged >70 years. CLINICALTRIALS. GOV REGISTRATION: NCT02555813. AUSTRIAN NIS REGISTRY: NIS005071.

Novel p53-dependent anticancer strategy by targeting iron signaling and BNIP3L-induced mitophagy.

This study identifies BNIP3L as the key regulator of p53-dependent cell death mechanism in colon cancer cells targeted by the novel gallium based anticancer drug, KP46. KP46 specifically accumulated into mitochondria where it caused p53-dependent morphological and functional damage impairing mitochondrial dynamics and bioenergetics. Furthermore, competing with iron for cellular uptake, KP46 lowered the intracellular labile iron pools and intracellular heme. Accordingly, p53 accumulated in the nucleus where it activated its transcriptional target BNIP3L, a BH3 only domain protein with functions in apoptosis and mitophagy. Upregulated BNIP3L sensitized the mitochondrial permeability transition and strongly induced PARKIN-mediated mitochondrial clearance and cellular vacuolization. Downregulation of BNIP3L entirely rescued cell viability caused by exposure of KP46 for 24 hours, confirming that early induced cell death was regulated by BNIP3L. Altogether, targeting BNIP3L in wild-type p53 colon cancer cells is a novel anticancer strategy activating iron depletion signaling and the mitophagy-related cell death pathway.

Comparative solution equilibrium studies of anticancer gallium(III) complexes of 8-hydroxyquinoline and hydroxy(thio)pyrone ligands.

The stoichiometry and stability constants of the Ga(III) complexes of 8-hydroxyquinoline (HQ), 8-hydroxyquinoline-5-sulfonate (HQS), maltol, thiomaltol, allomaltol and thioallomaltol were determined by means of pH-potentiometry, UV-vis spectrophotometry, spectrofluorometry and (1)H NMR spectroscopy in aqueous solution. Spectrofluorometry was used to determine the stability constants of the Ga(III)-HQ species in water. Formation of [GaL](2+), [GaL(2)](+) and [GaL(3)] complexes was found and the Ga(III) binding ability of the ligands followed the order: thioallomaltol

Targeting the DNA-topoisomerase complex in a double-strike approach with a topoisomerase inhibiting moiety and covalent DNA binder.

Ru(II)(arene)-flavonoids with high in vitro antitumour activity were synthesised. These compounds are capable of inhibiting human topoisomerase IIα and binding covalently to DNA.

Fluorescence properties and cellular distribution of the investigational anticancer drug triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) and its zinc(II) complex.

Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), which entered several phase I and II clinical trials as an antitumor chemotherapeutic agent, was found to possess intrinsic fluorescence properties (lambda(ex) = 360 nm), which enabled us to monitor the uptake and intracellular distribution in living human cancer cells by fluorescence microscopy.

Impact of metal coordination on cytotoxicity of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (triapine) and novel insights into terminal dimethylation.

The first metal complexes of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine) were synthesized. Triapine was prepared by a novel three-step procedure in 64% overall yield. In addition, a series of related ligands, namely, 2-formylpyridine thiosemicarbazone, 2-acetylpyridine thiosemicarbazone, 2-pyridineformamide thiosemicarbazone, and their N(4)-dimethylated derivatives (including the N(4)-dimethylated analogue of Triapine) were prepared, along with their corresponding gallium(III) and iron(III) complexes with the general formula [M(L)(2)](+), where HL is the respective thiosemicarbazone. The compounds were characterized by elemental analysis, (1)H and (13)C NMR, IR and UV-vis spectroscopies, mass spectrometry, and cyclic voltammetry. In addition, Triapine and its iron(III) and gallium(III) complexes were studied by X-ray crystallography. All ligands and complexes were tested for their in vitro antiproliferative activity in two human cancer cell lines (41M and SK-BR-3), and structure-activity relationships were established. In general, the coordination to gallium(III) increased the cytotoxicity while the iron(III) complexes show reduced cytotoxic activity compared to the metal-free thiosemicarbazones. Selected compounds were investigated for the capacity of inhibiting ribonucleotide reductase by incorporation of (3)H-cytidine into DNA.

Chilling and cultivar type affect the diversity of bacterial endophytes colonizing sweet pepper (Capsicum anuum L.).

A climate chamber experiment was conducted to assay the effect of low temperatures (chilling) on the diversity of bacteria colonizing the endospheres of two thermophilic sweet pepper (Capsicum anuum L.) cultivars, Milder Spiral and Ziegenhorn Bello. Structural diversity was analyzed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and by the generation of 16S rRNA gene libraries to determine dominant community members in T-RFLP profiles. Cultivable community members colonizing lines Milder Spiral and Ziegenhorn Bello were identified by 16S rRNA gene analysis. T-RFLP profiles and 16S rRNA gene libraries revealed a high heterogeneity of community composition due to chilling and suggested further the existence of cultivar-specific communities. The majority of isolates obtained from the cultivar Milder Spiral were assigned as high-G+C Gram-positive bacteria (Microbacterium sp., Micrococcus sp., Rhodococcus sp.) and Firmicutes (Staphylococcus sp.). Of the isolated endophytes obtained from cultivar Zeigenhorn Bello, 93% were affiliated with Staphylococcus aureus and Bacillus sp. (Firmicutes). The experimental set-up was suited to demonstrate that chilling and cultivar type can influence the diversity of bacterial endophytes colonizing sweet pepper. We propose additional chilling experiments to investigate the effect of chilling on functional, plant-beneficial abilities of bacterial endophytes associated with low-temperature-sensitive crops, such as sweet pepper.