Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells

Overexpression of SIRT1, a NAD⁺-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC₅₀ of 1, 10 and 0.5 μM, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells.     

Sodium-coupled electrogenic transport of pyroglutamate (5-oxoproline) via SLC5A8, a monocarboxylate transporter

Pyroglutamate, also known as 5-oxoproline, is a structural analog of proline. This amino acid derivative is a byproduct of glutathione metabolism, and is reabsorbed efficiently in kidney by Na⁺-coupled transport mechanisms. Previous studies have focused on potential participation of amino acid transport systems in renal reabsorption of this compound. Here we show that it is not the amino acid transport systems but instead the Na⁺-coupled monocarboxylate transporter SLC5A8 that plays a predominant role in this reabsorptive process. Expression of cloned human and mouse SLC5A8 in mammalian cells induces Na⁺-dependent transport of pyroglutamate that is inhibitable by various SLC5A8 substrates. SLC5A8-mediated transport of pyroglutamate is saturable with a Michaelis constant of 0.36 ± 0.04 mM. Na⁺-activation of the transport process exhibits sigmoidal kinetics with a Hill coefficient of 1.8 ± 0.4, indicating involvement of more than one Na⁺ in the activation process. Expression of SLC5A8 in Xenopuslaevis oocytes induces Na⁺-dependent inward currents in the presence of pyroglutamate under voltage-clamp conditions. The concentration of pyroglutamate necessary for induction of half-maximal current is 0.19 ± 0.01 mM. The Na⁺-activation kinetics is sigmoidal with a Hill coefficient of 2.3 ± 0.2. Ibuprofen, a blocker of SLC5A8, suppressed pyroglutamate-induced currents in SLC5A8-expressing oocytes; the concentration of the blocker necessary for causing half-maximal inhibition is 14 ± 1 μM. The involvement of SLC5A8 can be demonstrated in rabbit renal brush border membrane vesicles by showing that the Na⁺-dependent uptake of pyroglutamate in these vesicles is inhibitable by known substrates of SLC5A8. The Na⁺ gradient-driven pyroglutamate uptake was stimulated by an inside-negative K⁺ diffusion potential induced by valinomycin, showing that the uptake process is electrogenic.     

Cytotoxicity, intracellular distribution and uptake of doxorubicin and doxorubicin coupled to cell-penetrating peptides in different cell lines: A comparative study

One of the major obstacles which are opposed to the success of anticancer treatment is the cell resistance that generally develops after administration of commonly used drugs. In this study, we try to overcome the tumour cell resistance of doxorubicin (Dox) by developing a cell-penetrating peptide (CPP)-anticancer drug conjugate in aim to enhance its intracellular delivery and that its therapeutic effects. For this purpose, two cell-penetrating peptides, penetratin (pene) and tat, derived from the HIV-1 TAT protein, were chemically conjugated to Dox. The cytotoxicity, intracellular distribution and uptake were accessed in CHO cells (Chinese Hamster Ovarian carcinoma cells), HUVEC (Human Umbilical Vein Endothelial Cells), differentiated NG108.15 neuronal cell and breast cancer cells MCF7drug-sensitive or MDA-MB 231 drug-resistant cell lines. The conjugates showed different cell killing activity and intracellular distribution pattern by comparison to Dox as assessed respectively by MTT-based colorimetric cellular cytotoxicity assay, confocal fluorescence microscopy and FACS analysis. After treatment with 3 μM with Dox-CPPs for 2 h, pene increase the Dox cytotoxicity by 7.19-fold in CHO cells, by 11.53-fold in HUVEC cells and by 4.87-fold in MDA-MB 231 cells. However, cytotoxicity was decreased in NG108.15 cells and MCF7. Our CPPs-Dox conjugate proves the validity of CPPs for the cytoplasmic delivery of therapeutically useful molecules and also a valuable strategy to overcome drug resistance.     

In vitro inhibition of fatty acid synthase by 1,2,3,4,6-penta-O-galloyl-β-d-glucose plays a vital role in anti-tumour activity

1,2,3,4,6-Penta-O-galloyl-β-d-glucose (PGG) inhibits glioma cancer U251 cells, more strongly than MDA-MB-231 and U87 cells. In addition, PGG is transported across cancer cell membrane to further down-regulate FAS and activate caspase-3 in MDA-MB-231 cells. Compared with other FAS inhibitors, including catechin gallate and morin, PGG involves a higher reversible fast-binding inhibition with half-inhibitory concentration value (IC₅₀) of 1.16 μM and an irreversible slow-binding inhibition, i.e. saturation kinetics with a dissociation constant of 0.59 μM and a limiting rate constant of 0.16 min^−l. The major reacting site of PGG is on the β-ketoacyl reduction domain of FAS. PGG exhibits different types of inhibitions against the three substrates in the FAS overall reaction. The higher concentrations of PGG tested (higher than 20 μM) clearly altered the secondary structure of FAS by increasing the α-helix and induced a redshift in the FAS spectra. In addition, only PGG concentrations higher than 20 μM resulted in FAS precipitation.     

ABL Tyrosine Kinase Inhibition Variable Effects on the Invasive Properties of Different Triple Negative Breast Cancer Cell Lines

The non-receptor tyrosine kinase ABL drives myeloid progenitor expansion in human chronic myeloid leukemia. ABL inhibition by the tyrosine kinase inhibitor nilotinib is a first-line treatment for this disease. Recently, ABL has also been implicated in the transforming properties of solid tumors, including triple negative (TN) breast cancer. TN breast cancers are highly metastatic and several cell lines derived from these tumors display high invasive activity in vitro. This feature is associated with the activation of actin-rich membrane structures called invadopodia that promote extracellular matrix degradation. Here, we investigated nilotinib effect on the invasive and migratory properties of different TN breast cancer cell lines. Nilotinib decreased both matrix degradation and invasion in the TN breast cancer cell lines MDA-MB 231 and MDA-MB 468. However, and unexpectedly, nilotinib increased by two-fold the invasive properties of the TN breast cancer cell line BT-549 and of Src-transformed fibroblasts. Both display much higher levels of ABL kinase activity compared to MDA-MB 231. Similar effects were obtained by siRNA-mediated down-regulation of ABL expression, confirming ABL central role in this process. ABL anti-tumor effect in BT-549 cells and Src-transformed fibroblasts was not dependent on EGF secretion, as recently reported in neck and squamous carcinoma cells. Rather, we identified the TRIO-RAC1 axis as an important downstream element of ABL activity in these cancer cells. In conclusion, the observation that TN breast cancer cell lines respond differently to ABL inhibitors could have implications for future therapies.     

Characterization of a plasma membrane-resident albumin-binding protein associated with the proliferation of estrogen-target,serum-sensitive cells

Estrogens control the proliferation of their target cells through a receptor-mediated pathway. Recently presented evidence suggests that estradiol cancels the proliferative inhibition exerted by human albumin (HA) and recombinant human albumin (rHA) on estrogen-target serum-sensitive cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a plasma membrane estrogen receptor (mER) and a plasma membrane albumin-binding protein (mABP). Direct evidence confirming the presence of mERalpha in MCF7 cells has recently been presented. Herein, we now show that Western blot analysis of purified T47D membrane proteins with the C542 ERalpha specific monoclonal antibody also revealed specific, multiple M(r) mERs (67, 110, and 130k M(r)). In addition, Western blot analysis with an ABP antiserum revealed a potential 60k M(r) ABP in both MCF7 and T47D plasma membrane extracts. No such evidence was observed in similar extracts from ER-negative, serum-insensitive MDA-MB231 cells. Ligand blot analysis of similar plasma membrane extracts with bovine serum albumin confirmed the presence of a 60k M(r) ABP in MCF7 and T47D cells; again, no such evidence was observed in comparable extracts from MDA-MB231 cells. Fluorescence and confocal microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific membrane ABP antigenic sites by immunocytochemistry. Serum-insensitive MDA-MB231 cells fixed and labeled similarly did not exhibit this mABP. These results suggest that the proposed mABP is expressed only in serum-sensitive estrogen-target cells and is not expressed in cells insensitive to the proliferative inhibition of HA and rHA. Also, the present data suggest that the proposed mABP may be the recognition mechanism by which both HA and rHA inhibit MCF7 and T47D cell proliferation.     

Artichoke polyphenols induce apoptosis and decrease the invasive potential of the human breast cancer cell line MDA-MB231

The human breast cancer cell line, estrogen receptor negative, MDA-MB231, was used to evaluate the antitumor effect of polyphenolic extracts from the edible part of artichokes (AEs). Treatment of cancer cells reduced cell viability and inhibited cell growth in a dose-dependent manner. Importantly, AEs did not have any effect on normal breast epithelial cell line, MCF10A. Chlorogenic acid (ChA), the most representative component of the polyphenolic fraction of artichoke, had no prominent effects on the cell death rate of MDA-MB231 cells. The addition of AEs to the cells, rather than ChA, triggered apoptosis via a mitochondrial and a death-receptor pathway, as shown by the activation of caspase-9 and caspase-8, respectively. Furthermore, an increase of the Bax:Bcl2 ratio and up-regulation of cyclin-dependent kinase inhibitor, p21(WAF1), crucial apoptotic players, were documented. According to our data on activation of caspase-9, a loss of mitochondrial transmembrane potential (Ψ(m)) was shown. Cell motility and invasion capabilities were remarkably inhibited by AEs-treatment in highly invasive MDA-MB231 cells. In addition, a significant decrease of proteolytic activity of metalloproteinase-2 protein (MMP-2), involved in degrading components of the extracellular matrix, was detected. Our findings indicate that AEs reduced cell viability, inhibited cell growth, triggered apoptotic mechanisms, and showed inhibitory properties against the invasive behavior of MDA-MB231 cancer cell line. Altogether, these data indicate the potential chemopreventive activity of artichoke polyphenolic extracts.     

Effects of α-zearalenol on the metabolome of two breast cancer cell lines by 1H-NMR approach

Introduction

Zearalenone (ZEN) is one of the most widely distributed toxins that contaminates many crops and foods. Its major metabolites are α-Zearalenol (α-zol) and β-Zearalenol. Previous studies showed that ZEN and α-zol have estrogenic properties and are able to induce growth promoting effect in breast tissues.

Objectivies

Considering that tumorigenesis is dependent on the reprogramming of cellular metabolism and that the evaluation of the cellular metabolome is useful to understand the metabolic changes that can occur during the cancer development and progression or after treatments, aim of our work is to study, for the first time, the effects of α-zol on the metabolomic profile of an estrogen positive breast cancer cell line, MCF-7, and of an estrogen negative breast cancer cell lines MDA-MB231.

Methods

Firstly, we tested the effects of α-zol on the cell viability after 24, 48 and 72 h of treatments with 10^?10, 10^?8 and 10^?6 M concentrations on breast cancer MCF-7 and MDA-MB231 cell lines in comparison to human non-cancerous breast MCF10A cell line. Then, we evaluated cell cycle progression, levels of reactive oxygen species (ROS) and the metabolomic profiling by ¹H-NMR approach on MCF-7 and MDA-MB231 before and after 72 h treatments. Principal component analysis was used to compare the obtained spectra.

Results

α-zol is resulted able to induce: (i) an increase of the cell viability on MCF-7 cells mainly after 72 h treatment, (ii) a slight decrease of the cell viability on MDA-MB231 cells, and (iii) an increase of cells in S phase of the cell cycle and of ROS only in MCF-7 cells. Moreover, the evaluation of metabolomics profile evidenced that after treatment with α-zol the levels of some metabolites increased in MCF-7 cells whereas decreased slightly in MDA-MB231 cells.

Conclusions

Our results showed that α-zol was able to increase the protein biosynthesis as well as the lipid metabolism in MCF-7 cells, and, hence, to induce an estrogen positive breast cancer progression.

     

Hybrid pharmacophore design and synthesis of isatin–benzothiazole analogs for their anti-breast cancer activity

A hybrid pharmacophore approach was used to design and synthesize isatin–benzothiazole analogs to examine their anti-breast cancer activity. The cytotoxicity of these compounds were determined using three different human breast tumor cell lines, MDA-MB231, MDA-MB468, MCF7, and two non-cancer breast epithelial cell lines, 184B5 and MCF10A. Although all compounds examined were quite effective on all the cancer cell lines examined, the compounds 4-bromo-1-diethylaminomethyl-1H-indole-2,3-dione (2l) and 4-chloro-1-dimethylaminomethyl-3-(6-methyl-benzothiazol-2-ylimino)-1,3-dihydro-indol-2-one (5e) emerged as the most active compounds of this series. Importantly, the cytotoxic effect of 2l was 10–15-fold higher on cancer than non-cancer cells, suggesting that this compound can be very effective for the control of breast cancer with low side effects. Since 2l showed effective cytotoxicity on MCF7 cells and arrested the cells at G2/M at a similar concentration, these two phenomena may be closely correlated. We conclude that the isatin-linked benzothiazole analog can serve as a prototype molecule for further development of a new class of anti-breast cancer agents.     

Synthesis and in vitro anti-proliferative effects of 3-(hetero)aryl substituted 3-[(prop-2-ynyloxy)(thiophen-2-yl)methyl]pyridine derivatives on various cancer cell lines

A series of 3-(hetero)aryl substituted 3-[(prop-2-ynyloxy)(thiophen-2-yl)methyl]pyridine derivatives were designed as potential anticancer agents. These compounds were conveniently prepared by using Pd/C–Cu mediated Sonogashira type coupling as a key step. Many of these compounds were found to be promising when tested for their in vitro anti-proliferative properties against six cancer cell lines. All these compounds were found to be selective towards the growth inhibition of cancer cells with IC₅₀ values in the range of 0.9–1.7 μM (against MDA-MB 231 and MCF7 cells), comparable to the known anticancer drug doxorubicin.     

Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane

Aquaporin-1 (AQP1) is an integral membrane protein that facilitates osmotic water transport across cell plasma membranes in epithelia and endothelia. AQP1 has no known specific interactions with cytoplasmic or membrane proteins, but its recovery in a detergent-insoluble membrane fraction has suggested possible raft association. We tracked the membrane diffusion of AQP1 molecules labeled with quantum dots at an engineered external epitope at frame rates up to 91 Hz and over times up to 6 min. In transfected COS-7 cells, >75% of AQP1 molecules diffused freely over ∼7 μm in 5 min, with diffusion coefficient, D_1-3 ∼ 9 × 10⁻¹⁰ cm²/s. In MDCK cells, ∼60% of AQP1 diffused freely, with D_1-3 ∼ 3 × 10⁻¹⁰ cm²/s. The determinants of AQP1 diffusion were investigated by measurements of AQP1 diffusion following skeletal disruption (latrunculin B), lipid/raft perturbations (cyclodextrin and sphingomyelinase), and bleb formation. We found that cytoskeletal disruption had no effect on AQP1 diffusion in the plasma membrane, but that diffusion was increased greater than fourfold in protein de-enriched blebs. Cholesterol depletion in MDCK cells greatly restricted AQP1 diffusion, consistent with the formation of a network of solid-like barriers in the membrane. These results establish the nature and determinants of AQP1 diffusion in cell plasma membranes and demonstrate long-range nonanomalous diffusion of AQP1, challenging the prevailing view of universally anomalous diffusion of integral membrane proteins, and providing evidence against the accumulation of AQP1 in lipid rafts.     

GPI/AMF inhibition blocks the development of the metastatic phenotype of mature multi-cellular tumor spheroids

Epithelial–mesenchymal transition (EMT) and cellular invasiveness are two pivotal processes for the development of metastatic tumor phenotypes. The metastatic profile of non-metastatic MCF-7 cells growing as multi-cellular tumor microspheroids (MCTSs) was analyzed by determining the contents of the EMT, invasive and migratory proteins, as well as their migration and invasiveness potential and capacity to secrete active cytokines such as the glucose phosphate isomerase/AMF (GPI/AMF). As for the control, the same analysis was also performed in MCF-7 and MDA-MB-231 (highly metastatic, MDA) monolayer cells, and in stage IIIB and IV human metastatic breast biopsies. The proliferative cell layers (PRL) of mature MCF-7 MCTSs, MDA monolayer cells and metastatic biopsies exhibited increased cellular contents (2–15 times) of EMT (β-catenin, SNAIL), migratory (vimentin, cytokeratin, and fibronectin) and invasive (MMP-1, VEGF) proteins versus MCF-7 monolayer cells, quiescent cell layers of mature MCF-7 MCTS and non-metastatic breast biopsies. The increase in metastatic proteins correlated with substantially elevated cellular abilities for migration (18-times) and invasiveness (13-times) and with the higher level (6-times) of the cytokine GPI/AMF in the extracellular medium of PRL, as compared to MCF-7 monolayer cells. Interestingly, the addition of the GPI/AMF inhibitors erythrose-4-phosphate or 6-phosphogluconate at micromolar doses significantly decreased its extracellular activity (> 80%), with a concomitant diminution in the metastatic protein content and migratory tumor cell capacity, and with no inhibitory effect on tumor lactate production or toxicity on 3T3 mouse fibroblasts. The present findings provide new insights into the discovery of metabolic inhibitors to be used as complementary therapy against metastatic and aggressive tumors.     

Single-layer centrifugation through colloid selects improved quality of epididymal cat sperm

The objectives were to determine the: 1) extent of epithelial and red blood cell contamination in epididymal cat sperm samples recovered by the cutting method; 2) efficacy of simple washing, single-layer centrifugation (SLC), and swim-up for selecting epididymal cat sperm; and 3) effects of freezing and thawing on cat sperm selected by various techniques. Ten unit samples were studied; each contained sperm from the cauda epididymides of four cats (total, ∼200 × 10⁶ sperm) and was equally allocated into four treatments: 1) simple washing, 2) single-layer centrifugation through colloid prior to cryopreservation (SLC-PC), 3) single-layer centrifugation through colloid after cryopreservation (SLC-AC), and 4) swim-up. Centrifugation (300 × g for 20 min) was done for all methods. The SLC-PC had a better recovery rate than the SLC-AC and swim-up methods (mean ± SD of 16.4 ± 8.7, 10.7 ± 8.9, and 2.3 ± 1.7%, respectively; P < 0.05). The SLC-PC, SLC-AC and swim-up samples contained less red blood cell contamination than simple washed samples (0.02 ± 0.01, 0.02 ± 0.04, 0.03 ± 0.04, and 0.44 ± 0.22 × 10⁶ cells/mL, respectively; P < 0.05). Although the proportion of sperm with head abnormalities did not differ among selection methods (P > 0.05), SLC-PC yielded the highest percentage of sperm with normal midpieces and tails (P < 0.05), due to the lowest proportion of coiled tails (P < 0.05). Furthermore, the SLC-PC was as effective as swim-up in removing sperm with proximal droplets, and selecting motile sperm, as well as those with intact membranes and DNA (P > 0.05). In conclusion, both SLC-PC and swim-up improved the quality of epididymal cat sperm, including better morphology, membrane and DNA integrity, and removal of cellular contamination. However, SLC had a better sperm recovery rate than swim-up.     

Indole-3-carbinol-induced death in cancer cells involves EGFR downregulation and is exacerbated in a 3D environment

Indole-3-carbinol (I3C) is a promising anticancer dietary compound, which inhibits breast cancer in animal models. The objective of the current study was to characterize I3C-induced cell death in a panel of human breast tumorigenic cells (MCF7, MDA-MB-468, MDA-MB-231 and HBL100) in comparison with normal fibroblasts. Since epithelial cells are protected from cell death by a three-dimensional environment, 3D cell culture (collagen I gel and spheroids) was employed to investigate susceptibility to I3C. Cell viability in the presence of 256 μM I3C, a concentration close to the physiologically achievable range, was in the order fibroblasts = HBL100>MDA-MB-231>MCF7>MDA-MB-468 in monolayer culture. However, 3D culture conditions increased the susceptibility of MCF7 and MDA-MB-468 cancer cells towards I3C. I3C induced cell death in breast cancer MCF7, MDA-MB-468 and MDA-MB–231 cells via the mitochondrial apoptotic pathway. I3C significantly reduced levels of epidermal growth factor receptor (EGFR) in MDA-MB-468 after 6 h and in MDA-MB-231 and HBL100 cells after 30 h. Downregulation of EGFR in MDA-MB468 and MDA-MB-231 cells using an EGFR inhibitor resulted in apoptosis. EGFR modulation using EGF or an EGFR inhibitor markedly influenced viability and response to I3C in MDA-MB-468 cells in 3D conditions. EGFR expression was modulated by 3D conditions. Therefore, I3C-induced EGFR reduction in these cells is likely to be responsible for I3C-induced apoptosis.     

Oxidative and reductive pathways of estrogens in hormone responsive and non-responsive human breast cancer cells in vitro

In order to measure the formation and degradation rates of estradiol by human breast cancer cells, after assessing the biochemical basis of hormone responsiveness and growth response to estrogens, we considered both responsive, estrogen receptor (ER) positive, and non-responsive, ER-negative, breast cancer cell lines, i.e. MCF7, ZR75-1 and MDA-MB231. To this end, we employed a novel “intact cell” approach which allows us, after 24 h incubation, to analyze several enzyme activities in sequence, concurrently with the monitoring of labeled precursor degradation. Our investigations led to the following evidence: (a) the reductive activity of the 17β-hydroxysteroid oxoreductase (17β-HSOR) appears to be higher than the oxidative only in responsive, ER-rich MCF7 and ZR75-1 cells, as also previously observed by others; (b) this activity is, on the contrary, much lower in MDA-MB231 cells and other unresponsive, ER-poor breast cancer cell lines; (c) conversely, the oxidative activity shows an opposite pattern, being limited in MCF7 and ZR75-1 cells and much higher in MDA-MB231 cells. Overall, a 17β-HSOR reductive pathway prevails in both MCF7 and ZR75-1 cells, whilst the oxidative pathway is prevalent in MDA-MB231 cells, leading to a large formation of estrone that is no further metabolized, at least in the experimental conditions used. Our results may provide a likely explanation of previous data on the different estrogen content of breast tumor tissues.     

Evaluation of in vitro cytotoxicity and hepatotoxicity of platinum(II) and palladium(II) oxalato complexes with adenine derivatives as carrier ligands

In vitro antitumour activity of the [Pt(ox)(L_n)₂] (1-7) and [Pd(ox)(L_n)₂] (8-14) oxalato (ox) complexes involving N6-benzyl-9-isopropyladenine-based N-donor carrier ligands (L_n) against ovarian carcinoma (A2780), cisplatin resistant ovarian carcinoma (A2780cis), malignant melanoma (G-361), lung carcinoma (A549), cervix epitheloid carcinoma (HeLa), breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines was studied. Some of the tested complexes were even several times more cytotoxic as compared with cisplatin employed as a positive control. The improved cytotoxic effect was demonstrated for the platinum(II) complexes 3 (IC₅₀ = 3.2 ± 1.0 μM and 3.2 ± 0.6 μM) and 5 (IC₅₀ = 4.0 ± 1.0 μM and 4.1 ± 1.4 μM) against A2780 and A2780cis, as compared with 11.5 ± 1.6 μM, and 30.3 ± 6.1 μM determined for cisplatin, respectively. The significant in vitro cytotoxicity against MCF7 (IC₅₀ = 8.2 ± 3.8 μM for 12) and A2780 (IC₅₀ = 5.4 ± 1.2 μM for 14) was evaluated for the palladium(II) oxalato complexes, which again exceeded cisplatin, whose IC₅₀ equalled 19.6 ± 4.3 μM against the MCF7 cells. Selected complexes were also screened for their in vitro cytotoxic effect in primary cultures of human hepatocytes and they were found to be non-hepatotoxic.     

Protein C inhibitor regulates both cathepsin L activity and cell-mediated tumor cell migration

Background

Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation including thrombin and activated protein C. However, the physiological role of PCI remains under investigation. The cysteine protease, cathepsin L, has a role in many physiological processes including cardiovascular diseases, blood vessel remodeling, and cancer.

Methods and results

We found that PCI inhibits cathepsin L with an inhibition rate (k₂) of 3.0 × 10⁵ M⁻¹ s⁻¹. Whereas, the PCI P1 mutant (R354A) inhibits cathepsin L at rates similar to wild-type PCI, mutating the P2 residue results in a slight decrease in the rate of inhibition. We then assessed the effect of PCI and cathepsin L on the migration of human breast cancer (MDA-MB-231) cells. Cathepsin L was expressed in both the cell lysates and conditioned media of MDA-MB-231 cells. Wound-induced and transwell migration of MDA-MB-231 cells was inhibited by exogenously administered wtPCI and PCI P1 but not PCI P14 mutant. In addition, migration of MDA-MB-231 cells expressing wtPCI was significantly decreased compared to non-expressing MDA-MB-231 cells or MDA-MB-231 cells expressing the PCI P14 mutant. Downregulation of cathepsin L by either a specific cathepsin L inhibitor or siRNA technology also resulted in a decrease in the migration of MDA-MB-231 cells.

Conclusions

Overall, our data show that PCI regulates tumor cell migration partly by inhibiting cathepsin L.

General significance

Consequently, inhibiting cathepsin L by serpins like PCI may be a new pathway of regulating hemostasis, cardiovascular and metastatic diseases.     

Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus

The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (K_d, ∼10⁻¹¹ M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R² = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R² = 0.834). These results suggest that the BV–KinExA method described here provides a suitably accurate K_d evaluation of mAbs against proteins on the cell surface.     

Flavokawain A Induces Apoptosis in MCF-7 and MDA-MB231 and Inhibits the Metastatic Process In Vitro

Introduction

The kava-kava plant (Piper methsyticum) is traditionally known as the pacific elixir by the pacific islanders for its role in a wide range of biological activities. The extract of the roots of this plant contains a variety of interesting molecules including Flavokawain A and this molecule is known to have anti-cancer properties. Breast cancer is still one of the leading diagnosed cancers in women today. The metastatic process is also very pertinent in the progression of tumorigenesis.

Methods

MCF-7 and MDA-MB231 cells were treated with several concentrations of FKA. The apoptotic analysis was done through the MTT assay, BrdU assay, Annexin V analysis, cell cycle analysis, JC-1 mitochondrial dye, AO/PI dual staining, caspase 8/9 fluorometric assay, quantitative real time PCR and western blot. For the metastatic assays, the in vitro scratch assay, trans-well migration/invasion assay, HUVEC tube formation assay, ex vivo rat aortic ring assay, quantitative real time PCR and western blot were employed.

Results

We have investigated the effects of FKA on the apoptotic and metastatic process in two breast cancer cell lines. FKA induces apoptosis in both MCF-7 and MDA-MB231 in a dose dependent manner through the intrinsic mitochondrial pathway. Additionally, FKA selectively induces a G2/M arrest in the cell cycle machinery of MDA-MB231 and G1 arrest in MCF-7. This suggests that FKA''s anti-cancer activity is dependent on the p53 status. Moreover, FKA also halted the migration and invasion process in MDA-MB231. The similar effects can be seen in the inhibition of the angiogenesis process as well.

Conclusions

FKA managed to induce apoptosis and inhibit the metastatic process in two breast cancer cell lines, in vitro. Overall, FKA may serve as a promising candidate in the search of a new anti-cancer drug especially in halting the metastatic process but further in vivo evidence is needed.