Generation of 3D Fibroblast-Derived Extracellular Matrix and Analysis of Tumor Cell-Matrix Interactions and Signaling.

Fibroblasts are the major producers of the extracellular matrix and regulate its organization. Aberrant signaling in diseases such as fibrosis and cancer can impact the deposition of the matrix proteins, which can in turn act as an adhesion scaffold and signaling reservoir promoting disease progression. To study the composition and organization of the extracellular matrix as well as its interactions with (tumor) cells, this protocol describes the generation and analysis of 3D fibroblast-derived matrices and the investigation of (tumor) cells seeded onto the 3D scaffolds by immunofluorescent imaging and cell adhesion, colony formation, migration, and invasion/transmigration assays.

Ectopic expression of HNF4α in Het1A cells induces an invasive phenotype.

Barrett's oesophagus (BO) is a pathological condition in which the squamous epithelium of the distal oesophagus is replaced by an intestinal-like columnar epithelium originating from the gastric cardia. Several somatic mutations contribute to the intestinal-like metaplasia. Once these have occurred in a single cell, it will be unable to expand further unless the altered cell can colonise the surrounding squamous epithelium of the oesophagus. The mechanisms by which this happens are still unknown. Here we have established an in vitro system for examining the competitive behaviour of two epithelia. We find that when an oesophageal epithelium model (Het1A cells) is confronted by an intestinal epithelium model (Caco-2 cells), the intestinal cells expand into the oesophageal domain. In this case the boundary involves overgrowth by the Caco-2 cells and the formation of isolated colonies. Two key transcription factors, normally involved in intestinal development, HNF4α and CDX2, are both expressed in BO. We examined the competitive ability of Het1A cells stably expressing HNF4α or CDX2 and placed in confrontation with unmodified Het1A cells. The key result is that stable expression of HNF4α, but not CDX2, increased the ability of the cells to migrate and push into the unmodified Het1A domain. In this situation the boundary between the cell types is a sharp one, as is normally seen in BO. The experiments were conducted using a variety of extracellular substrates, which all tended to increase the cell migration compared to uncoated plastic. These data provide evidence that HNF4α expression could have a potential role in the competitive spread of BO into the oesophagus as HNF4α increases the ability of cells to invade into the adjacent stratified squamous epithelium, thus enabling a single mutant cell eventually to generate a macroscopic patch of metaplasia.

Conductive Polymer-Coated 3D Printed Microneedles: Biocompatible Platforms for Minimally Invasive Biosensing Interfaces.

Conductive polymeric microneedle (MN) arrays as biointerface materials show promise for the minimally invasive monitoring of analytes in biodevices and wearables. There is increasing interest in microneedles as electrodes for biosensing, but efforts have been limited to metallic substrates, which lack biological stability and are associated with high manufacturing costs and laborious fabrication methods, which create translational barriers. In this work, additive manufacturing, which provides the user with design flexibility and upscale manufacturing, is employed to fabricate acrylic-based microneedle devices. These microneedle devices are used as platforms to produce intrinsically-conductive, polymer-based surfaces based on polypyrrole (PPy) and poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS). These entirely polymer-based solid microneedle arrays act as dry conductive electrodes while omitting the requirement of a metallic seed layer. Two distinct coating methods of 3D-printed solid microneedles, in situ polymerization and drop casting, enable conductive functionality. The microneedle arrays penetrate ex vivo porcine skin grafts without compromising conductivity or microneedle morphology and demonstrate coating durability over multiple penetration cycles. The non-cytotoxic nature of the conductive microneedles is evaluated using human fibroblast cells. The proposed fabrication strategy offers a compelling approach to manufacturing polymer-based conductive microneedle surfaces that can be further exploited as platforms for biosensing.

Phenanthroindolizidine Alkaloids Isolated from Tylophora ovata as Potent Inhibitors of Inflammation, Spheroid Growth, and Invasion of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC), representing the most aggressive form of breast cancer with currently no targeted therapy available, is characterized by an inflammatory and hypoxic tumor microenvironment. To date, a broad spectrum of anti-tumor activities has been reported for phenanthroindolizidine alkaloids (PAs), however, their mode of action in TNBC remains elusive. Thus, we investigated six naturally occurring PAs extracted from the plant Tylophora ovata: O-methyltylophorinidine (1) and its five derivatives tylophorinidine (2), tylophoridicine E (3), 2-demethoxytylophorine (4), tylophoridicine D (5), and anhydrodehydrotylophorinidine (6). In comparison to natural (1) and for more-in depth studies, we also utilized a sample of synthetic O-methyltylophorinidine (1s). Our results indicate a remarkably effective blockade of nuclear factor kappa B (NFκB) within 2 h for compounds (1) and (1s) (IC50 = 17.1 ± 2.0 nM and 3.3 ± 0.2 nM) that is different from its effect on cell viability within 24 h (IC50 = 13.6 ± 0.4 nM and 4.2 ± 1 nM). Furthermore, NFκB inhibition data for the additional five analogues indicate a structure-activity relationship (SAR). Mechanistically, NFκB is significantly blocked through the stabilization of its inhibitor protein kappa B alpha (IκBα) under normoxic as well as hypoxic conditions. To better mimic the TNBC microenvironment in vitro, we established a 3D co-culture by combining the human TNBC cell line MDA-MB-231 with primary murine cancer-associated fibroblasts (CAF) and type I collagen. Compound (1) demonstrates superiority against the therapeutic gold standard paclitaxel by diminishing spheroid growth by 40% at 100 nM. The anti-proliferative effect of (1s) is distinct from paclitaxel in that it arrests the cell cycle at the G0/G1 state, thereby mediating a time-dependent delay in cell cycle progression. Furthermore, (1s) inhibited invasion of TNBC monoculture spheroids into a matrigel®-based environment at 10 nM. In conclusion, PAs serve as promising agents with presumably multiple target sites to combat inflammatory and hypoxia-driven cancer, such as TNBC, with a different mode of action than the currently applied chemotherapeutic drugs.

Targeting the Hippo/YAP/TAZ signalling pathway: Novel opportunities for therapeutic interventions into skin cancers.

Skin cancers are by far the most frequently diagnosed human cancers. The closely related transcriptional co-regulator proteins YAP and TAZ (WWTR1) have emerged as important drivers of tumour initiation, progression and metastasis in melanoma and non-melanoma skin cancers. YAP/TAZ serve as an essential signalling hub by integrating signals from multiple upstream pathways. In this review, we summarize the roles of YAP/TAZ in skin physiology and tumorigenesis and discuss recent efforts of therapeutic interventions that target YAP/TAZ in in both preclinical and clinical settings, as well as their prospects for use as skin cancer treatments.

Cancer-Associated Fibroblasts Suppress CD8+ T-cell Infiltration and Confer Resistance to Immune-Checkpoint Blockade.

Immune-checkpoint blockade (ICB) promotes antitumor immune responses and can result in durable patient benefit. However, response rates in breast cancer patients remain modest, stimulating efforts to discover novel treatment options. Cancer-associated fibroblasts (CAF) represent a major component of the breast tumor microenvironment and have known immunosuppressive functions in addition to their well-established roles in directly promoting tumor growth and metastasis. Here we utilized paired syngeneic mouse mammary carcinoma models to show that CAF abundance is associated with insensitivity to combination αCTLA4 and αPD-L1 ICB. CAF-rich tumors exhibited an immunologically cold tumor microenvironment, with transcriptomic, flow cytometric, and quantitative histopathologic analyses demonstrating a relationship between CAF density and a CD8+ T-cell-excluded tumor phenotype. The CAF receptor Endo180 (Mrc2) is predominantly expressed on myofibroblastic CAFs, and its genetic deletion depleted a subset of αSMA-expressing CAFs and impaired tumor progression in vivo. The addition of wild-type, but not Endo180-deficient, CAFs in coimplantation studies restricted CD8+ T-cell intratumoral infiltration, and tumors in Endo180 knockout mice exhibited increased CD8+ T-cell infiltration and enhanced sensitivity to ICB compared with tumors in wild-type mice. Clinically, in a trial of melanoma patients, high MRC2 mRNA levels in tumors were associated with a poor response to αPD-1 therapy, highlighting the potential benefits of therapeutically targeting a specific CAF subpopulation in breast and other CAF-rich cancers to improve clinical responses to immunotherapy. SIGNIFICANCE: Paired syngeneic models help unravel the interplay between CAF and tumor immune evasion, highlighting the benefits of targeting fibroblast subpopulations to improve clinical responses to immunotherapy.

Impairment of a distinct cancer-associated fibroblast population limits tumour growth and metastasis.

Profiling studies have revealed considerable phenotypic heterogeneity in cancer-associated fibroblasts (CAFs) present within the tumour microenvironment, however, functional characterisation of different CAF subsets is hampered by the lack of specific markers defining these populations. Here we show that genetic deletion of the Endo180 (MRC2) receptor, predominantly expressed by a population of matrix-remodelling CAFs, profoundly limits tumour growth and metastasis; effects that can be recapitulated in 3D co-culture assays. This impairment results from a CAF-intrinsic contractility defect and reduced CAF viability, which coupled with the lack of phenotype in the normal mouse, demonstrates that upregulated Endo180 expression by a specific, potentially targetable CAF subset is required to generate a supportive tumour microenvironment. Further, characterisation of a tumour subline selected via serial in vivo passage for its ability to overcome these stromal defects provides important insight into, how tumour cells adapt to a non-activated stroma in the early stages of metastatic colonisation.

Preclinical studies on metal based anticancer drugs as enabled by integrated metallomics and metabolomics.

Resistance development is a major obstacle for platinum-based chemotherapy, with the anticancer drug oxaliplatin being no exception. Acquired resistance is often associated with altered drug accumulation. In this work we introduce a novel -omics workflow enabling the parallel study of platinum drug uptake and its distribution between nucleus/protein and small molecule fraction along with metabolic changes after different treatment time points. This integrated metallomics/metabolomics approach is facilitated by a tailored sample preparation workflow suitable for preclinical studies on adherent cancer cell models. Inductively coupled plasma mass spectrometry monitors the platinum drug, while the metabolomics tool-set is provided by hydrophilic interaction liquid chromatography combined with high-resolution Orbitrap mass spectrometry. The implemented method covers biochemical key pathways of cancer cell metabolism as shown by a panel of >130 metabolite standards. Furthermore, the addition of yeast-based 13C-enriched internal standards upon extraction enabled a novel targeted/untargeted analysis strategy. In this study we used our method to compare an oxaliplatin sensitive human colon cancer cell line (HCT116) and its corresponding resistant model. In the acquired oxaliplatin resistant cells distinct differences in oxaliplatin accumulation correlated with differences in metabolomic rearrangements. Using this multi-omics approach for platinum-treated samples facilitates the generation of novel hypotheses regarding the susceptibility and resistance towards oxaliplatin.

Metabolic adaptability in metastatic breast cancer by AKR1B10-dependent balancing of glycolysis and fatty acid oxidation.

The different stages of the metastatic cascade present distinct metabolic challenges to tumour cells and an altered tumour metabolism associated with successful metastatic colonisation provides a therapeutic vulnerability in disseminated disease. We identify the aldo-keto reductase AKR1B10 as a metastasis enhancer that has little impact on primary tumour growth or dissemination but promotes effective tumour growth in secondary sites and, in human disease, is associated with an increased risk of distant metastatic relapse. AKR1B10High tumour cells have reduced glycolytic capacity and dependency on glucose as fuel source but increased utilisation of fatty acid oxidation. Conversely, in both 3D tumour spheroid assays and in vivo metastasis assays, inhibition of fatty acid oxidation blocks AKR1B10High-enhanced metastatic colonisation with no impact on AKR1B10Low cells. Finally, mechanistic analysis supports a model in which AKR1B10 serves to limit the toxic side effects of oxidative stress thereby sustaining fatty acid oxidation in metabolically challenging metastatic environments.

Generation and characterisation of two D2A1 mammary cancer sublines to model spontaneous and experimental metastasis in a syngeneic BALB/c host.

Studying the complex mechanisms underlying breast cancer metastasis and therapy response necessitates relevant in vivo models, particularly syngeneic models with an intact immune system. Two syngeneic spontaneously metastatic sublines, D2A1-m1 and D2A1-m2, were generated from the poorly metastasising BALB/c-derived D2A1 cell line by serial in vivo passaging. In vivo and in vitro analyses revealed distinct and shared characteristics of the metastatic D2A1-m1 and D2A1-m2 sublines. In particular, D2A1-m1 cells are more aggressive in experimental metastasis assays, while D2A1-m2 cells are more efficient at disseminating from the primary tumour in spontaneous metastasis assays. Surprisingly, classical metastasis-associated in vitro phenotypes, such as enhanced proliferation, migration and invasion, are reduced in the sublines compared to the parental cell line. Further, evasion of immune control cannot fully explain their enhanced metastatic properties. By contrast, both sublines show increased resistance to apoptosis when cultured in non-adherent conditions and, for the D2A1-m2 subline, increased 3D tumour spheroid growth. Moreover, the enhanced spontaneous metastatic phenotype of the D2A1-m2 subline is associated with an increased ability to recruit an activated tumour stroma. The metastatic D2A1-m1 and D2A1-m2 cell lines provide additional syngeneic models for investigating the different steps of the metastatic cascade and thereby represent valuable tools for breast cancer researchers. Finally, this study highlights that morphology and cell behaviour in 2D cell-based assays cannot be used as a reliable predictor of metastatic behaviour in vivo.

Comparative studies of oxaliplatin-based platinum(iv) complexes in different in vitro and in vivo tumor models.

Using platinum(iv) prodrugs of clinically established platinum(ii) compounds is a strategy to overcome side effects and acquired resistances. We studied four oxaliplatin-derived platinum(iv) complexes with varying axial ligands in various in vitro and in vivo settings. The ability to interfere with DNA (pUC19) in the presence and absence of a reducing agent (ascorbic acid) was investigated in cell-free experiments. Cytotoxicity was compared under normoxic and hypoxic conditions in monolayer cultures and multicellular spheroids of colon carcinoma cell lines. Effects on the cell cycle were investigated by flow cytometry, and the capacity of inducing apoptosis was confirmed by flow cytometry and Western blotting. The anti-cancer activity of one complex was studied in vivo in immunodeficient and immunocompetent mice, and the platinum levels in various organs and the tumor after treatment were quantified. The results demonstrate that modification of the axial ligands can improve the cytotoxic potency. The complexes are able to interfere with plasmid DNA, which is enhanced by co-incubation with a reducing agent, and cause cell cycle perturbations. At higher concentrations, they induce apoptosis, but generate only low levels of reactive oxygen species. Two of the complexes increase the life span of leukemia (L1210) bearing mice, and one showed effects similar to oxaliplatin in a CT26 solid tumor model, despite the low platinum levels in the tumor. As in the case of oxaliplatin, activity in the latter model depends on an intact immune system. These findings show new perspectives for the development of platinum(iv) prodrugs of the anticancer agent oxaliplatin, combining bioreductive properties and immunogenic aspects.

Calpain-mediated integrin deregulation as a novel mode of action for the anticancer gallium compound KP46.

On the basis of enhanced tumor accumulation and bone affinity, gallium compounds are under development as anticancer and antimetastatic agents. In this study, we analyzed molecular targets of one of the lead anticancer gallium complexes [KP46, Tris(8-quinolinolato)gallium(III)] focusing on colon and lung cancer. Within a few hours, KP46 treatment at low micromolar concentrations induced cell body contraction and loss of adhesion followed by prompt cell decomposition. This rapid KP46-induced cell death lacked classic apoptotic features and was insensitive toward a pan-caspase inhibitor. Surprisingly, however, it was accompanied by upregulation of proapoptotic Bcl-2 family members. Furthermore, a Bax- but not a p53-knockout HCT-116 subline exhibited significant KP46 resistance. Rapid KP46-induced detachment was accompanied by downregulation of focal adhesion proteins, including several integrin subunits. Loss of integrin-ß1 and talin plasma membrane localization corresponded to reduced binding of RGD (Arg-Gly-Asp) peptides to KP46-treated cells. Accordingly, KP46-induced cell death and destabilization of integrins were enhanced by culture on collagen type I, a major integrin ligand. In contrast, KP46-mediated adhesion defects were partially rescued by Mg(2+) ions, promoting integrin-mediated cell adhesion. Focal adhesion dynamics are regulated by calpains via cleavage of multiple cell adhesion molecules. Cotreatment with the cell-permeable calpain inhibitor PD150606 diminished KP46-mediated integrin destabilization and rapid cell death induction. KP46 treatment distinctly inhibited HCT-116 colon cancer xenograft in vivo by causing reduced integrin plasma membrane localization, tissue disintegration, and intense tumor necrosis. This study identifies integrin deregulation via a calpain-mediated mechanism as a novel mode of action for the anticancer gallium compound KP46.

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.

The ruthenium compound KP1339 potentiates the anticancer activity of sorafenib in vitro and in vivo.

KP1339 is a promising ruthenium-based anticancer compound in early clinical development. This study aimed to test the effects of KP1339 on the in vitro and in vivo activity of the multi-kinase inhibitor sorafenib, the current standard first-line therapy for advanced hepatoma. Anticancer activity of the parental compounds as compared to the drug combination was tested against a panel of cancer cell lines with a focus on hepatoma. Combination of KP1339 with sorafenib induced in the majority of all cases distinctly synergistic effects, comprising both sorafenib-resistant as well as sorafenib-responsive cell models. Several mechanisms were found to underlie these multifaceted synergistic activities. Firstly, co-exposure induced significantly enhanced accumulation levels of both drugs resulting in enhanced apoptosis induction. Secondly, sorafenib blocked KP1339-mediated activation of P38 signalling representing a protective response against the ruthenium drug. In addition, sorafenib treatment also abrogated KP1339-induced G2/M arrest but resulted in check point-independent DNA-synthesis block and a complete loss of the mitotic cell populations. The activity of the KP1339/sorafenib combination was evaluated in the Hep3B hepatoma xenograft. KP1339 monotherapy led to a 2.4-fold increase in life span and, thus, was superior to sorafenib, which induced a 1.9-fold prolonged survival. The combined therapy further enhanced the mean survival by 3.9-fold. Synergistic activity was also observed in the VM-1 melanoma xenograft harbouring an activating braf mutation. Together, our data indicate that the combination of KP1339 with sorafenib displays promising activity in vitro and in vivo especially against human hepatoma models.

Synergistic anticancer activity of arsenic trioxide with erlotinib is based on inhibition of EGFR-mediated DNA double-strand break repair.

Arsenic trioxide (ATO), one of the oldest remedies used in traditional medicine, was recently rediscovered as an anticancer drug and approved for treatment of relapsed acute promyelocytic leukemia. However, its activity against nonhematologic cancers is rather limited so far. Here, we show that inhibition of ATO-mediated EGF receptor (EGFR) activation can be used to potently sensitize diverse solid cancer types against ATO. Thus, combination of ATO and the EGFR inhibitor erlotinib exerted synergistic activity against multiple cancer cell lines. Subsequent analyses revealed that this effect was based on the blockade of ATO-induced EGFR phosphorylation leading to more pronounced G2-M arrest as well as enhanced and more rapid induction of apoptosis. Comparable ATO-sensitizing effects were also found with PI3K/AKT and mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitors, suggesting an essential role of the EGFR-mediated downstream signaling pathway in cancer cell protection against ATO. H2AX staining and comet assay revealed that erlotinib significantly increases ATO-induced DNA double-strand breaks (DSB) well in accordance with a role of the EGFR signaling axis in DNA damage repair. Indeed, EGFR inhibition led to downregulation of several DNA DSB repair proteins such as Rad51 and Rad50 as well as reduced phosphorylation of BRCA1. Finally, the combination treatment of ATO and erlotinib was also distinctly superior to both monotreatments against the notoriously therapy-resistant human A549 lung cancer and the orthotopic p31 mesothelioma xenograft model in vivo. In conclusion, this study suggests that combination of ATO and EGFR inhibitors is a promising therapeutic strategy against various solid tumors harboring wild-type EGFR.

Organometallic anticancer complexes of lapachol: metal centre-dependent formation of reactive oxygen species and correlation with cytotoxicity.

Organometallic Ru(II), Os(II) and Rh(III) complexes of lapachol induce apoptosis in human tumour cell lines in the low µM range by a mode of action involving oxidative stress, especially in the case of the ruthenium compound.

Influence of extracellular pH on the cytotoxicity, cellular accumulation, and DNA interaction of novel pH-sensitive 2-aminoalcoholatoplatinum(II) complexes.

Extracellular acidity is a frequent pathophysiological condition of solid tumors offering possibilities for improving the tumor selectivity of molecular therapy. This might be accomplished by prodrugs with low systemic toxicity, attaining their full antitumor potency only under acidic conditions, such as bis(2-aminoalcoholato-κ(2)N,O)platinum(II) complexes that are activated by protonation of alcoholato oxygen, resulting in cleavage of platinum-oxygen bonds. In this work, we examined whether the pH dependency of such compounds is reflected in differential biological activity in vitro. In particular, the pH dependence of cytotoxicity, cellular accumulation, DNA platination, GMP binding, effects on DNA secondary structure, cell cycle alterations, and induction of apoptosis was investigated. Enhanced cytotoxicity of five of these complexes in non-small-cell lung cancer (A549) and colon carcinoma (HT-29) cells at pH 6.0 in comparison with pH 7.4 was confirmed: 50 % growth inhibition concentrations ranged from 42 to 214 µM in A549 cells and from 35 to 87 µM in HT-29 cells at pH 7.4 and decreased at pH 6.0 to 11-50 and 7.3-25 µM, respectively. The effects induced by all five pH-sensitive compounds involve increased 5'-GMP binding, cellular accumulation, and DNA platination as well as stronger effects on DNA secondary structure at pH 6.0 than at pH 7.4. As exemplified by treatment of A549 cells with a 2-amino-4-methyl-1-pentanolato complex, induction of apoptosis is enhanced at pH 6.5. These results confirm the increased reactivity and in vitro activity of these compounds under slightly acidic conditions, encouraging further evaluation of ring-closed aminoalcoholatoplatinum(II) derivatives in solid tumors in vivo.

Impact of terminal dimethylation on the resistance profile of α-N-heterocyclic thiosemicarbazones.

Triapine is an α-N-heterocyclic thiosemicarbazone with promising anticancer activity against hematologic malignancies but widely ineffective against solid tumor types in clinical trials. The anticancer activity of thiosemicarbazones can be dramatically increased by terminal dimethylation. KP1089 is a gallium compound containing two terminal dimethylated thiosemicarbazone ligands. To gain insights on the vulnerability of this highly active terminal dimethylated thiosemicarbazone to drug resistance mechanisms, a new cell model with acquired resistance against the lead compound KP1089 was established. Subsequent genomic analyses (arrayCGH and FISH) revealed amplification of the ABCC1 gene on double minute chromosomal DNA in KP1089-resistant cells as well as overexpression of ABCC1 and ABCG2 on the protein level. KP1089 was further confirmed as a substrate of ABCC1 and ABCG2 but not of ABCB1 using a panel of ABC transporter-overexpressing cell models as well as ABC transporter inhibitors. Moreover, glutathione depletion strongly enhanced KP1089 activity, although no glutathione conjugate formation by glutathione-S-transferase was observed. Thus, a co-transport of KP1089 together with glutathione is suggested. Finally, a panel of thiosemicarbazone derivatives was tested on the new KP1089-resistant cell line. Notably, KP1089-resistant cells were not cross-resistant against thiosemicarbazones lacking terminal dimethylation (e.g. Triapine) which are less active than KP1089. This suggests that terminal dimethylation of thiosemicarbazones - linked with distinctly enhanced anticancer activity - leads to altered resistance profiles compared to classical thiosemicarbazones making this compound class of interest for further (pre)clinical evaluation.

Anticancer activity of methyl-substituted oxaliplatin analogs.

Oxaliplatin is successfully used in systemic cancer therapy. However, resistance development and severe adverse effects are limiting factors for curative cancer treatment with oxaliplatin. The purpose of this study was to comparatively investigate in vitro and in vivo anticancer properties as well as the adverse effects of two methyl-substituted enantiomerically pure oxaliplatin analogs [[(1R,2R,4R)-4-methyl-1,2-cyclohexanediamine] oxalatoplatinum(II) (KP1537), and [(1R,2R,4S)-4-methyl-1,2-cyclohexanediamine]oxalatoplatinum(II) (KP1691)] and to evaluate the impact of stereoisomerism. Although the novel oxaliplatin analogs demonstrated in multiple aspects activities comparable with those of the parental compound, several key differences were discovered. The analogs were characterized by reduced vulnerability to resistance mechanisms such as p53 mutations, reduced dependence on immunogenic cell death induction, and distinctly attenuated adverse effects including weight loss and cold hyperalgesia. Stereoisomerism of the substituted methyl group had a complex and in some aspects even contradictory impact on drug accumulation and anticancer activity both in vitro and in vivo. To summarize, methyl-substituted oxaliplatin analogs harbor improved therapeutic characteristics including significantly reduced adverse effects. Hence, they might be promising metal-based anticancer drug candidates for further (pre)clinical evaluation.

Targeted proteolysis of plectin isoform 1a accounts for hemidesmosome dysfunction in mice mimicking the dominant skin blistering disease EBS-Ogna.

Autosomal recessive mutations in the cytolinker protein plectin account for the multisystem disorders epidermolysis bullosa simplex (EBS) associated with muscular dystrophy (EBS-MD), pyloric atresia (EBS-PA), and congenital myasthenia (EBS-CMS). In contrast, a dominant missense mutation leads to the disease EBS-Ogna, manifesting exclusively as skin fragility. We have exploited this trait to study the molecular basis of hemidesmosome failure in EBS-Ogna and to reveal the contribution of plectin to hemidesmosome homeostasis. We generated EBS-Ogna knock-in mice mimicking the human phenotype and show that blistering reflects insufficient protein levels of the hemidesmosome-associated plectin isoform 1a. We found that plectin 1a, in contrast to plectin 1c, the major isoform expressed in epidermal keratinocytes, is proteolytically degraded, supporting the notion that degradation of hemidesmosome-anchored plectin is spatially controlled. Using recombinant proteins, we show that the mutation renders plectin's 190-nm-long coiled-coil rod domain more vulnerable to cleavage by calpains and other proteases activated in the epidermis but not in skeletal muscle. Accordingly, treatment of cultured EBS-Ogna keratinocytes as well as of EBS-Ogna mouse skin with calpain inhibitors resulted in increased plectin 1a protein expression levels. Moreover, we report that plectin's rod domain forms dimeric structures that can further associate laterally into remarkably stable (paracrystalline) polymers. We propose focal self-association of plectin molecules as a novel mechanism contributing to hemidesmosome homeostasis and stabilization.