Effective Drug Concentration and Selectivity Depends on Fraction of Primitive Cells.

Poor efficiency of chemotherapeutics in the eradication of Cancer Stem Cells (CSCs) has been driving the search for more active and specific compounds. In this work, we show how cell density-dependent stage culture profiles can be used in drug development workflows to achieve more robust drug activity (IC50 and EC50) results. Using flow cytometry and light microscopy, we characterized the cytological stage profiles of the HL-60-, A-549-, and HEK-293-derived sublines with a focus on their primitive cell content. We then used a range of cytotoxic substances-C-123, bortezomib, idarubicin, C-1305, doxorubicin, DMSO, and ethanol-to highlight typical density-related issues accompanying drug activity determination. We also showed that drug EC50 and selectivity indices normalized to primitive cell content are more accurate activity measurements. We tested our approach by calculating the corrected selectivity index of a novel chemotherapeutic candidate, C-123. Overall, our study highlights the usefulness of accounting for primitive cell fractions in the assessment of drug efficiency.

Interactions between FGFR2 and RSK2-implications for breast cancer prognosis.

We have previously demonstrated that fibroblast growth factor receptor 2 (FGFR2) activates ribosomal s6 kinase 2 (RSK2) in mammary epithelial cells and that this pathway promotes in vitro cell growth and migration. Potential clinical significance of FGFR2 and RSK2 association has never been investigated. Herein, we have undertaken an evaluation of a possible relationship between FGFR2/RSK2 interdependence and disease outcome in breast cancer (BCa) patients. The clinical analysis was complemented by an in vitro investigation of an involvement of RSK2 in the regulation of FGFR2 function. Primary tumour samples from 152 stage I-III BCa patients were examined for FGFR2 and RSK2 gene and protein expression. FGFR2 showed a positive correlation with RSK2 at both protein (p = 0.003) and messenger RNA (mRNA) (p = 0.001) levels. Lack of both FGFR2 and activated RSK (RSK-P) significantly correlated with better disease-free survival (DFS) (p = 0.01). Patients with tumours displaying immunoreactivity for either or both FGFR2 and RSK-P had 4.89-fold higher risk of recurrence when compared to the FGFR2/RSK-P-negative subgroup. FGFR2-RSK2 interactions were verified by co-immunoprecipitation and internalization assays in HB2 mammary epithelial cell line (characterized by high endogenous FGFR2 and RSK2 expression). In vitro analyses revealed that FGFR2 and RSK2 formed an indirect complex and that activated RSK exerted a significant impact on fibroblast growth factor 2 (FGF2)-triggered internalization of FGFR2. Our results suggest that the FGFR2-RSK2 signalling pathway is involved in pathophysiology of BCa and evaluation of FGFR2/RSK-P expression may be useful in disease prognostication.

Phosphorylation of RSK2 at Tyr529 by FGFR2-p38 enhances human mammary epithelial cells migration.

The members of p90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases are downstream effectors of MAPK/ERK pathway that regulate diverse cellular processes including cell growth, proliferation and survival. In carcinogenesis, RSKs are thought to modulate cell motility, invasion and metastasis. Herein, we have studied an involvement of RSKs in FGF2/FGFR2-driven behaviours of mammary epithelial and breast cancer cells. We found that both silencing and inhibiting of FGFR2 attenuated phosphorylation of RSKs, whereas FGFR2 overexpression and/or its stimulation with FGF2 enhanced RSKs activity. Moreover, treatment with ERK, Src and p38 inhibitors revealed that p38 kinase acts as an upstream RSK2 regulator. We demonstrate for the first time that in FGF2/FGFR2 signalling, p38 but not MEK/ERK, indirectly activated RSK2 at Tyr529, which facilitated phosphorylation of its other residues (Thr359/Ser363, Thr573 and Ser380). In contrast to FGF2-triggered signalling, inhibition of p38 in the EGF pathway affected only RSK2-Tyr529, without any impact on the remaining RSK phosphorylation sites. p38-mediated phosphorylation of RSK2-Tyr529 was crucial for the transactivation of residues located at kinase C-terminal domain and linker-region, specifically, in the FGF2/FGFR2 signalling pathway. Furthermore, we show that FGF2 promoted anchorage-independent cell proliferation, formation of focal adhesions and cell migration, which was effectively abolished by treatment with RSKs inhibitor (FMK). These indicate that RSK2 activity is indispensable for FGF2/FGFR2-mediated cellular effects. Our findings identified a new FGF2/FGFR2-p38-RSK2 pathway, which may play a significant role in the pathogenesis and progression of breast cancer and, hence, may present a novel therapeutic target in the treatment of FGFR2-expressing tumours.

[Aspects of progesterone receptor (PR) activity regulation - impact on breast cancer progression]. / Aspekty regulacji aktywnosci receptora progesteronu (PR) - znaczenie w progresji raka gruczolu piersiowego.

Progesterone receptor (PR) and its specific ligand play a key role in development and physiology of mammary gland. The role of PR in initiation and progression of breast carcinoma (BCa) is unquestionable, although molecular mechanism of PR action is complex and not fully understood. It is known that increased risk of breast cancer is associated with progestin-based (synthetic ligands of progesterone) hormonal contraception or hormone replacement therapies. It is estimated that ER/PR-positive tumours represent approximately 50-70% of all BCa cases, and the loss of PR is associated with resistance to hormonal therapy and increased tumour invasiveness. In classical, genomic signalling pathway cytoplasmic PR, following ligand binding, translocates to the nucleus and regulates expression of genes with the PRE sequence. PR is also involved in a large number of alternative, non-genomic signalling cascades, e.g. PR is able to activate MAPK and PI3K/AKT pathways, which leads to regulation of gene expression. The cross-talk between PR and Growth Factors Receptors (GFR) results in progesterone-independent activation of PR as well as PR-regulated GFR expression and activation. Growth factors signalling promotes formation of a pool of hypersensitive PR responsive to even very low ligand concentration. Transcriptional activity of PR as well as its dynamic impact on processes such as cell migration and adhesion are crucial for BCa progression. Further studies of multifaceted mechanisms of PR action may contribute to new PR-targeting therapeutic strategies for breast cancer patients.

Mechanism of cytotoxic action of perfluorinated acids. III. Disturbance in Ca²+ homeostasis.

The global distribution of perfluorinated acids (PFAs) in industry and in household is well known. Their increasing environmental occurrence and biomagnification in the living organisms have drawn growing interests in efforts to describe precisely the mechanisms of action in vitro and in vivo. Our previous investigations widely described lipophilicity-dependent cytotoxicity of PFAs as well as the effect of perfluorination of carbon chain on depolarization of plasma membrane potential, acidification or mitochondrial dysfunctions. In this study we presented in dose- and time-dependent manner the impact of PFAs on calcium homeostasis in HCT116 cells. Comparative analysis of cytosolic [Ca²+](c) and mitochondrial calcium [Ca²+](m) carried out by flow cytometry revealed distinct uptake of calcium into mitochondria in correlation to increasing lipophilicity of PFAs. Massive accumulation of [Ca²+](m) was not accompanied by equivalent loss of [Ca²+](c). Indeed, moderate changes of [Ca²+](c) were observed after incubation with 400 µM PFDoDA reaching 29.83% and 49.17% decrease at 4th and 72nd hour, respectively. At the same time, mitochondrial calcium uptake increased from 2- to more than 4-fold comparing with non-treated cells. Incubation with non-fluorinated decanoic acid (DA) did not cause any changes in calcium homeostasis. Presented data show that PFAs-induced perturbations in calcium distribution seem to be a missing link related to mitochondria dysfunction playing a crucial role in determination of apoptotic cell death. Complete scheme for the mechanism of cytotoxic action of PFAs has been included.

Triazoloacridone C-1305 abrogates the restriction checkpoint in cells lacking functional p53 and promotes their accumulation in the G2/M phase of the cell cycle.

Triazoloacridone C-1305, a new topoisomerase II inhibitor, exhibits potent cytostatic activity toward various tumours under in vitro and in vivo conditions. Interestingly, mouse cells lacking poly(ADP-ribose) polymerase-1 are much more sensitive to C-1305 than their normal counterparts. In the present study we tested the hypothesis that the functional status of p53 in tumour cells might have an impact on the efficiency of C-1305 in experiments with both p53-deficient human HL-60 promyelocytic leukemia cells and human MCF-7 breast cancer cells harboring a functional p53 pathway. Exposure of both cancer cell lines to C-1305 reduced the number of viable cells in a time- and concentration-dependent manner. Remarkably, however, HL-60 cells were much more strongly affected than MCF-7 cells. Measurements of DNA concentrations in single cells revealed that C-1305 arrested the tested cancer cells at the G/M transition. Analysis of the cell cycle and apoptosis regulators revealed that C-1305 strongly elevated phosphorylation of CDK1 at the inhibitory sites (Thr14/Tyr15) in HL-60 cells. Furthermore, C-1305 increased phosphorylation of pRb protein and CDK2 at Thr160 in HL60 cells, but not in MCF-7 cells. These observations suggest that C-1305 abrogates the restriction checkpoint and promotes G1/S transition in cells lacking functional p53.

p38 Mediates Resistance to FGFR Inhibition in Non-Small Cell Lung Cancer.

FGFR signalling is one of the most prominent pathways involved in cell growth and development as well as cancer progression. FGFR1 amplification occurs in approximately 20% of all squamous cell lung carcinomas (SCC), a predominant subtype of non-small cell lung carcinoma (NSCLC), indicating FGFR as a potential target for the new anti-cancer treatment. However, acquired resistance to this type of therapies remains a serious clinical challenge. Here, we investigated the NSCLC cell lines response and potential mechanism of acquired resistance to novel selective FGFR inhibitor CPL304110. We found that despite significant genomic differences between CPL304110-sensitive cell lines, their resistant variants were characterised by upregulated p38 expression/phosphorylation, as well as enhanced expression of genes involved in MAPK signalling. We revealed that p38 inhibition restored sensitivity to CPL304110 in these cells. Moreover, the overexpression of this kinase in parental cells led to impaired response to FGFR inhibition, thus confirming that p38 MAPK is a driver of resistance to a novel FGFR inhibitor. Taken together, our results provide an insight into the potential direction for NSCLC targeted therapy.

MET-Pyk2 Axis Mediates Acquired Resistance to FGFR Inhibition in Cancer Cells.

Deregulation of fibroblast growth factor receptors (FGFRs) signaling, as a result of FGFR amplification, chromosomal translocation, or mutations, is involved in both initiation and progression of a wide range of human cancers. Clinical data demonstrating the dependence of cancer cells on FGFRs signaling clearly indicate these receptors as the molecular targets of anti-cancer therapies. Despite the increasing number of tyrosine kinase inhibitors (TKIs) being investigated in clinical trials, acquired resistance to these drugs poses a serious therapeutic problem. In this study, we focused on a novel pan-FGFR inhibitor-CPL304110, currently being investigated in phase I clinical trials in adults with advanced solid malignancies. We analyzed the sensitivity of 17 cell lines derived from cancers with aberrant FGFR signaling, i.e. non-small cell lung cancer, gastric and bladder cancer to CPL304110. In order to explore the mechanism of acquired resistance to this FGFR inhibitor, we developed from sensitive cell lines their variants resistant to CPL304110. Herein, for the first time we revealed that the process of acquired resistance to the novel FGFR inhibitor was associated with increased expression of MET in lung, gastric, and bladder cancer cells. Overexpression of MET in NCI-H1703, SNU-16, RT-112 cells as well as treatment with HGF resulted in the impaired response to inhibition of FGFR activity. Moreover, we demonstrated that cells with acquired resistance to FGFR inhibitor as well as cells overexpressing MET displayed enhanced migratory abilities what was accompanied with increased levels of Pyk2 expression. Importantly, inhibition of both MET and Pyk2 activity restored sensitivity to FGFR inhibition in these cells. Our results demonstrate that the HGF/MET-Pyk2 signaling axis confers resistance to the novel FGFR inhibitor, and this mechanism is common for lung, gastric, and bladder cancer cells. Our study suggests that targeting of MET/Pyk2 could be an approach to overcome resistance to FGFR inhibition.

Structural determinants of imidazoacridinones facilitating antitumor activity are crucial for substrate recognition by ABCG2.

Symadex is the lead acridine compound of a novel class of imidazoacridinones (IAs) currently undergoing phase II clinical trials for the treatment of various cancers. Recently, we have shown that Symadex is extruded by ABCG2-overexpressing lung cancer A549/K1.5 cells, thereby resulting in a marked resistance to certain IAs. To identify the IA residues essential for substrate recognition by ABCG2, we here explored the ability of ABCG2 to extrude and confer resistance to a series of 23 IAs differing at defined residue(s) surrounding their common 10-azaanthracene structure. Taking advantage of the inherent fluorescent properties of IAs, ABCG2-dependent efflux and drug resistance were determined in A549/K1.5 cells using flow cytometry in the presence or absence of fumitremorgin C, a specific ABCG2 transport inhibitor. We find that a hydroxyl group at one of the R1, R2, or R3 positions in the proximal IA ring was essential for ABCG2-mediated efflux and consequent IA resistance. Moreover, elongation of the common distal aliphatic side chain attenuated ABCG2-dependent efflux, thereby resulting in the retention of parental cell sensitivity. Hence, the current study offers novel molecular insight into the structural determinants that facilitate ABCG2-mediated drug efflux and consequent drug resistance using a unique platform of fluorescent IAs. Moreover, these results establish that the IA determinants mediating cytotoxicity are precisely those that facilitate ABCG2-dependent drug efflux and IA resistance. The possible clinical implications for the future design of novel acridines that overcome ABCG2-dependent multidrug resistance are discussed.

Tenascin C interacts with ecto-5'-nucleotidase (eN) and regulates adenosine generation in cancer cells.

Tenascin C is expressed in invasive human solid tumors; however its specific role in cancer biology remains obscure. Previously, we have found that ecto-5'-nucleotidase (eN) is a marker of ER (-) breast carcinoma and elevated expression correlates with invasive mesenchymal cell phenotype. To investigate for the potential relationship between eN and protein components of the extracellular matrix (ECM) we measured adenosine generation from AMP in cells incubated with soluble ECM proteins. We found that tenascin C was the only ECM component that strongly inhibited ecto-5'-nucleotidase (eN) activity in situ and adenosine generation from AMP (75% inhibition, p < 0.01). The inhibition was comparable to that induced by concanavalin A, a well-defined and strong inhibitor of eN. Resin immobilized tenascin C, but not collagen, and only weakly fibronectin, specifically and quantitatively bound cell-extracted eN. We further developed breast cancer cell line with reduced eN expression and tested changes in cell adhesion on different ECM. Breast cancer cells expressing reduced eN attached 56% weaker (p < 0.05) to immobilized tenascin C. This difference was not detected with other ECM proteins. Finally, control breast cancer cells migrated slower on tenascin C when compared with clone with reduced eN expression. These data suggest that eN is a novel and specific receptor for tenascin C and that the interaction between these proteins may influence cell adhesion and migration and also lead to decreased generation of local adenosine.

Mechanism of cytotoxic action of perfluorinated acids. I. alteration in plasma membrane potential and intracellular pH level.

Perfluorinated (aliphatic) acids (PFAs) and congeners have many applications in various industrial fields and household for decades. Years later they have been detected in wildlife and this has spurred interest in environmental occurrence as well as influencing living organisms. PFAs were established as peroxisome proliferators and hepatocarcinogens. Amphipatic structure suggests that they may alter cell membrane potential (mbDeltaPsi) and/or induce changes in cytosolic pH (pHi). The aim of this study was to examine the correlation between changes of above parameters and PFAs structure (CF(6)-CF(12)) in human colon carcinoma HCT116 cells. mbDeltaPsi and pHi were measured by flow cytometry using fluorescence polarization of the plasma membrane probe 3,3'-dipentyloxacarbocyanine (DiOC(5)(3)) and fluorescein diacetate (FDA), respectively. Dose- and time-dependent manner analysis revealed relatively fast depolarization of plasma membrane and acidification of cytosol both positively correlated with fluorocarbon chain length. mbDeltaPsi depletion after 4 h of incubation reached 8.01% and 30.08% for 50 muM PFOA and 50 muM PFDoDA, respectively. Prolonged treatment (72 h) led to dramatic dissipation of membrane potential up to 21.65% and 51.29% and strong acidification to pHi level at 6.92 and 6.03 at the presence of above compounds, respectively. The data demonstrate that PFAs can alter plasma membrane protonotrophy with the mode dependent on the compound hydrophobicity.

Mechanism of cytotoxic action of perfluorinated acids II. Disruption of mitochondrial bioenergetics.

PFAs and derivatives due to perfect technological properties are broadly applied in industry and consumer goods, and in consequence widely disseminated, environmentally bioaccumulative and found at ppb level in human serum. Earlier we revealed that in vitro cytotoxicity increases with chain length (CF(6)-CF(14)). The compounds dissipate plasma membrane potential and acidify of cytosol. Here we determine whether there is an association between the protonophoric uncoupling of respiration and disruption of bioenergetics caused by CF(6)-CF(12) on HCT116 cell apoptosis. Again the effects were stronger for longer molecules. Incubation of cells with CF(10) stimulated time-dependent generation of reactive oxygen species, opening of mitochondrial permeability transition (MPT) pore, release of cytochrome c, activation of caspases and depletion of intracellular level of ATP occurring in intrinsic pathway of apoptosis. Incubation with decanoic acid (DA) did not lead to mitochondrial dysfunctions neither to cell cycle disturbances. Synchronized removal of the phosphorylated state of Akt, ERK1/2 and PKCdelta/theta kinases by CF(10) suggests presence of concerted action to uninhibit Bad protein activation and a cascade of intrinsic pathway of apoptosis. Blocking MPT pore by cyclosporin A (CsA) led to a reduction of mitochondrial potential dissipation (mtDeltaPsi). Such cells neither showed cytochrome c release nor the downstream activation of caspase-9 and caspase-3. Our results confirm that mitochondria play a crucial role in perfluorochemicals induced apoptosis by releasing apoptotic signals through MPT pore.

Induction of the multixenobiotic/multidrug resistance system in various cell lines in response to perfluorinated carboxylic acids.

The multixenobiotic resistance (closely related to multidrug resistance) system controls transport across the plasma membrane as a defense against toxic molecules. Multixenobiotic resistance system consists of an efflux pump, ABCB1 (also named P-glycoprotein, P-gp), and/or a molecule of the ABCC family (also named multiple resistance associated protein, MRP). ABCB1 is able to increase efflux of many low-molecular foreign molecules. Measuring system induction may be used as a biomarker of cell/organism exposure to foreign substances. Various established cell lines were tested for constitutive and induced multixenobiotic resistance proteins by Western blotting immunodetection. The pumping function was indirectly assayed with Rhodamine B by visualization of cell fluorescence in the presence of verapamil. Changes in ABC proteins were measured by flow cytometry after exposition to various perfluorinated carboxylic acids. MCF7 and HeLa cells were found to contain the highest constitutive level of both ABCB1 and ABCC1. HEK293 exhibited much less ABCB1 and no activity of pumping out Rhodamine B. The pumping activity was found to be related to the amount of the cell-type specific 170 kDa ABCB1 protein. An 8-day exposure to 10(-4) M perfluorononanoic acid resulted in about 2-2.5-fold increase of ABCB1 level. That was confirmed also for short times by flow cytometry of cells exposed to perfluorinated acids and its natural congeners. Both ABCB1- and ABCC1-related fluorescence increased along with the carbon chain in acids from C(6) up to C(9) and decreased for C(10). Measuring of multixenobiotic resistance changes in vitro induced by chemicals may be a convenient test for screening for their potential toxicity.

Progesterone impairs Herceptin effect on breast cancer cells.

Breast cancer (BCa) is the most common cancer affecting women worldwide. Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in ~20-25% of invasive ductal breast carcinomas and is associated with the more aggressive phenotype. Herceptin, a humanized antibody against HER2, is a standard therapy in HER2-overexpressing cases. Approximately one-third of patients relapse despite treatment. Therefore numerous studies have investigated the molecular mechanisms associated with Herceptin resistance. An interaction between HER2 signalling and steroid hormone receptor signalling pathways has been previously investigated, but the effect of this relationship on Herceptin resistance has never been studied. The present study analysed an impact of the steroid hormone, progesterone (PG), on Herceptin-dependent cell growth inhibition. Results indicated that Herceptin-inhibited proliferation of breast cancer cell lines overexpressing HER2 (BT474 and MCF/HER2) in 3D culture is abolished by PG. Furthermore, results demonstrated that PG led to the activation of HER2/HER3-mediated signalling. Moreover, PG treatment induced a shift of Herceptin-dependent cell cycle arrest in G1 phase towards S and G2 phases with concomitant upregulation of cyclin-dependent kinase 2 (CDK2) and downregulation of CDK inhibitor p27Kip1. These results demonstrate for the first time PG involvement in the failure of Herceptin treatment in vitro. The present observations suggest that cross-talk between PG- and HRG/HER2-initiated signalling pathways may lead to the acquisition of resistance to Herceptin in patients with BCa.

Cell Density-Dependent Cytological Stage Profile and Its Application for a Screen of Cytostatic Agents Active Toward Leukemic Stem Cells.

Proliferation and expansion of leukemia is driven by leukemic stem cells (LSCs). Multidrug resistance (MDR) of LSCs is one of the main reasons of failure and relapses in acute myeloid leukemia (AML) treatment. In this study, we show that maintaining HL-60 at low cell culture density or applying a 240-day treatment with anthrapyridazone (BS-121) increased the percentage of primitive cells, which include LSCs determining the overall stage profile. This change manifested in morphology, expression of both cell surface markers and redox-state proteins, as well as mitochondrial potential. Moreover, four sublines were generated, each with unique and characteristic stage profile and cytostatic sensitivity. Cell density-induced culture alterations (affecting stage profiles) were exploited in a screen of anthrapyridazones. Among the compound tested, C-123 was the most potent against primitive cell stages while generating relatively low amounts of reactive oxygen species (ROS). Furthermore, it had low toxicity in vivo and weakly affected blood morphology of healthy mice. The cell density-dependent stage profiles could be utilized in preliminary drug screens for activity against LSCs or in construction of patient-specific platforms to find drugs effective in case of AML relapse (drug extrapolation). The correlation between ROS generation in differentiated cells and toxic effect observed in HL-60 has a potential application in myelotoxicity predictions. The discovered properties of C-123 indicate its potential application in AML treatment, specifically in conditioned myeloablation preceding allogeneic transplantation and/or ex vivo treatment preceding autologous transplantation.

C421 allele-specific ABCG2 gene amplification confers resistance to the antitumor triazoloacridone C-1305 in human lung cancer cells.

The A421 ABCG2 genotype is a frequent polymorphism encoding the K141 transporter, which is associated with a significant decrease in transporter expression and function when compared to the wild type (wt) C421 allele encoding the Q141 ABCG2. Here we show that during the acquisition of resistance to the novel triazoloacridone antitumor agent C-1305 in lung cancer cells harboring a heterozygous C421A genotype, a marked C421 allele-specific ABCG2 gene amplification occurred. This monoallelic C421 ABCG2 gene amplification brought about the overexpression of both C421 ABCG2 mRNA and the transporter at the plasma membrane. This resulted in the lack of cellular drug accumulation due to increased efflux of both C1305 and C-1311, a fluorescent imidazoacridone homologue of C-1305, as well as marked resistance to these antitumor agents and to established ABCG2 substrates including mitoxantrone and SN-38. Consistently, the accumulation and sensitivity to these drugs were restored upon incubation with the potent and specific ABCG2 transport inhibitors Ko143 and fumitremorgin C. Moreover, upon transfection into HEK293 cells, the wt Q141 ABCG2 allele displayed a significantly decreased accumulation of C-1311 and increased resistance to C-1305, C-1311 and mitoxantrone, when compared to the K141 ABCG2 transfectant. Hence, the current study provides the first evidence that during the exposure to anticancer drugs, an allele-specific Q141 ABCG2 gene amplification occurs that confers a drug resistance advantage when compared to the K141 ABCG2. These findings have important implications for the selection and expansion of malignant anticancer drug resistant clones during chemotherapy with ABCG2 drugs.

Acid-catalyzed synthesis of oxathiolone fused chalcones. Comparison of their activity toward various microorganisms and human cancer cells line.

Substituted oxathiolone fused chalcones were prepared by condensation of 4-acetyl-5-methoxy-2-oxo-benz[1,3]oxathiole with benzaldehydes under acidic conditions. The compounds were tested for cytotoxic, antibacterial, antifungal and tuberculostatic activity. Three derivatives demonstrated weak activity against HeLa cells, two were slightly active against Micrococcus luteus and Staphylococcus aureus, and one was active against Mycobacterium tuberculosis H(37)Rv.

Analysis of structure-cytotoxicity in vitro relationship (SAR) for perfluorinated carboxylic acids.

Perfluorinated carboxylic acids (PFAs) represent derivatives of naturally occurring compounds and have been widely used in various industrial fields for decades. They are known to be environmentally persistent. Thus far numerous reports have been focused on reproductive toxicity of PFAs in animals but few studies have been carried out on toxicity towards human cells. Viability tests were performed here at varying time-exposures on C6-C18 PFAs with human colon carcinoma (HCT116) cells. These cells were found earlier as the most useful line for in vitro assays. A chain length-EC50 dependence has been clearly observed. Estimated values of EC50 decreased with elongation of fluorocarbon chain (PFHxA > PFHpA > PFOA > PFNA > PFDA > PFDoA > PFTeDA). Further elongation (C16 and C18) did not deepen the effect but even partially reversed it. The effect was intensified after longer exposure (72 h); at relatively low 40 microM PFTeDA, the viability decreased to approximately 50%. It seems that PFAs are not acutely toxic at the cellular level. Even so, however, they can trigger cell apoptosis, which is prominent in the case of myristic acid perfluorinated analogue.

Induction of unique structural changes in guanine-rich DNA regions by the triazoloacridone C-1305, a topoisomerase II inhibitor with antitumor activities.

We recently reported that the antitumor triazoloacridone, compound C-1305, is a topoisomerase II poison with unusual properties. In this study we characterize the DNA interactions of C-1305 in vitro, in comparison with other topoisomerase II inhibitors. Our results show that C-1305 binds to DNA by intercalation and possesses higher affinity for GC- than AT-DNA as revealed by surface plasmon resonance studies. Chemical probing with DEPC indicated that C-1305 induces structural perturbations in DNA regions with three adjacent guanine residues. Importantly, this effect was highly specific for C-1305 since none of the other 22 DNA interacting drugs tested was able to induce similar structural changes in DNA. Compound C-1305 induced stronger structural changes in guanine triplets at higher pH which suggested that protonation/deprotonation of the drug is important for this drug-specific effect. Molecular modeling analysis predicts that the zwitterionic form of C-1305 intercalates within the guanine triplet, resulting in widening of both DNA grooves and aligning of the triazole ring with the N7 atoms of guanines. Our results show that C-1305 binds to DNA and induces very specific and unusual structural changes in guanine triplets which likely plays an important role in the cytotoxic and antitumor activity of this unique compound.

Synthesis and biological evaluation of fluorinated N-benzoyl and N-phenylacetoyl derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate as steroid sulfatase inhibitors.

In the present work, we report convenient methods for the synthesis of 3-(4-aminophenyl)-coumarin-7-O-sulfamate derivatives N-acylated with fluorinated analogues of benzoic or phenylacetic acid as steroid sulfatase (STS) inhibitors. The design of these potential STS inhibitors was supported by molecular modeling techniques. Additionally, computational docking methods were used to determine the binding modes of the synthesized inhibitors and to identify potential interactions between inhibitors and amino acid residues located in the active site of STS. The inhibitory effects of the synthesized compounds were tested on STS isolated from human placenta and against estrogen receptor-(ER)-positive MCF-7 and T47D cells, as well as ER-negative MDA-MB-231 and SkBr3 cancer cell lines. In the course of our investigation, compounds 6c and 6j demonstrated the highest inhibitory effect in enzymatic STS assays, both with IC50 values of 0.18 µM (the IC50 value of coumarin-7-O-sulfamate is 1.38 µM, used as a reference). Compound 6j exhibited the highest potency against the MCF-7 and T47D cell lines (15.9 µM and 8.7 µM, respectively). The GI50 values of tamoxifen (used as a reference) were 6.8; 10.6; 15.1; 12.5 µM against MCF-7, T47D, MDA-MB-231 and SkBr3 cancer cell lines, respectively. Despite the slightly lower activity of compounds 1 and 2 (both in enzymatic and cell-based experiments) compared to 6g and 6j, analogues 1 and 2 proved to selectively inhibit the growth of ER- and PR-positive cell lines.