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Explore Talking Angela’s world and customize her fashion, hairstyle, makeup and home - all while playing addictively cute mini-games. With over 165 million downloads already don’t miss out on the fun! ADOPT BABY ANGELA Adopt Angela as your very own virtual pet and give her a fabulous life!

Help her grow into a stylish city kitty. From teeth brushing to clothes shopping – she’s all yours! TAKE CARE OF HER Make Angela your very own superstar! Nurture her, sing to her, feed her delicious treats. Just watch – she’ll become your new best friend!

CREATE COLORFUL MAKEUP Let your stunning sense of style shine through by giving Talking Angela a style make-over! Lipstick, eyeshadow, blush - customize to your heart's content! With dozens of different colors and endless creative freedom; you can really express yourself! EXPRESS YOUR FASHION FLAIR You’re fabulous, so make Angela fabulous too! Dress her in the latest fashions and the cutest costumes, from beautiful ballerina to punk ninja! Complete the look by giving her the perfect hairstyle too! With over a million different fashion combinations, you can create something truly unique!

PLAY MINI-GAMES Discover and play amazing new mini-games! From Happy Connect to Bubble Shooter - all your favorites are here and more are added all the time! AND MUCH, MUCH MORE. Unlock exclusive new outfits, level up, collect special stickers, customize her fabulous home all while she repeats everything you say - in classic Angela style! This app is PRIVO certified. The PRIVO safe harbor seal indicates Outfit7 has established COPPA compliant privacy practices to protect your child’s personal information. Our apps do not allow younger children to share their information.

So cute game.! I like it very much. The shoe collection is awesome. Dresses and outfits are so much.

Hairstyles are glamorous. This is a perfect game.😉 But I work so hard to go to new levels and when I finally come to sticker album and see my new pack of stickers I see all 5 stickers are duplicate I don't like it minimum 2 stickers should be duplicate. Please Pleased do anything for it.

This thing makes the game's repotation down. 😬😬 The spring hairstyle is the bestest hairstyle 🌹🌹 I like it's all colours but blue is the most glamorous 😘.

I think that this is a really fun game!!!!! It's not realistic because you don't put makeup on real cats and they don't stand on 2 legs or eat human food all day!!!! If you have allergies to cats and you want to see what it's like to have a cat this game is not a game that real cats do or its like based on cats but this is also a fun game because you can control the cat and in real life you can't control everything the cat does unless you are a mind controller!!! It's kind of educational too because it shows you all the things you need to do in life like eat,sleep,play,have different clothes on everyday and you also get to collect stuff and you get to change her hair!!!! It's just a really fun game and it's not a time waster getting this game IS WORTH IT💝💝. Hey Outfit or all of u readers out there!! I rated this a 5 star.

For all of you guys thinking this is not a safe game to play, just know that u are safe because it has been verified by Play Protect so it is safe and there is nothing to worry about. Anyway back to the game itself it's amazing!! Truly a piece of outstanding work and lots of effort put into it. I also like the dresses and the accessories but there is only one thing that bothers me. THE DUPLICATES! It gets on my nerves sometimes so that's my only complaint but it's still awesome!

So yea don't be scared to play it's verified and I suggest you play it because it's so awesome!! So cute game.! I like it very much. The shoe collection is awesome. Dresses and outfits are so much. Hairstyles are glamorous. This is a perfect game.😉 But I work so hard to go to new levels and when I finally come to sticker album and see my new pack of stickers I see all 5 stickers are duplicate I don't like it minimum 2 stickers should be duplicate.

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Please Pleased do anything for it. This thing makes the game's repotation down. 😬😬 The spring hairstyle is the bestest hairstyle 🌹🌹 I like it's all colours but blue is the most glamorous 😘. I think that this is a really fun game!!!!! It's not realistic because you don't put makeup on real cats and they don't stand on 2 legs or eat human food all day!!!! If you have allergies to cats and you want to see what it's like to have a cat this game is not a game that real cats do or its like based on cats but this is also a fun game because you can control the cat and in real life you can't control everything the cat does unless you are a mind controller!!!

It's kind of educational too because it shows you all the things you need to do in life like eat,sleep,play,have different clothes on everyday and you also get to collect stuff and you get to change her hair!!!! It's just a really fun game and it's not a time waster getting this game IS WORTH IT💝💝.

Hey Outfit or all of u readers out there!! I rated this a 5 star. For all of you guys thinking this is not a safe game to play, just know that u are safe because it has been verified by Play Protect so it is safe and there is nothing to worry about. Anyway back to the game itself it's amazing!! Truly a piece of outstanding work and lots of effort put into it. I also like the dresses and the accessories but there is only one thing that bothers me.

THE DUPLICATES! It gets on my nerves sometimes so that's my only complaint but it's still awesome! So yea don't be scared to play it's verified and I suggest you play it because it's so awesome!! I love the game my Talking Angela I like it very very much. 💝💝💝💝💝💝💝💝💝💝💝 I like the hair styles makeup dresses and the shoe collection playing this game even I want to become Angela but everything is so expensive. And when I reach next level I will get the same duplicate stickers I will reach so hardly to the next level.

I think so you have to change this and often it happens like talking Angela is not responding do you want to close it.It often happens I get irritated but now it is not happening like that I love the game my Talking Angela keep improving upgrading thank you for the game. 😫😫😫 i have hardly collected money 💰 for buying a dress 👗 but by mistake I clicked on an other object and it directly got purchased, there was no chance to confirm which irritated me a lot.

I mistakenly spent a lot of money on an unwanted item 😧🤑😲. And many items are very very expensive which are hard to collect Money for. So try to decrease ur prices.

The other thing is that when our kitty is 100 percent awake ⚡then also we can play very less games 🕹that is other thing that irritates. 😑😑😑😑😑😑IF U INPROVE THE GAME I WILL SURELY GIVE U FIVE STARS 🌟🌟🌟🌟🌟. So I think this game is very fascinating very educational and very funny cuz if Amanda was a baby like OMG what in the world that was so adorable this game is the best thing ever it's like I really love this game. Sorry if you hear some people yelling in the background I'm really sorry okay I am so so so so so so sorry I'm sorry if you had heard people yelling in the background that was pretty weird but just ignore them okay cuz I have no idea what's going on and they're just kind of bad so anyway just oh my God.

The process of cancer invasion is a multistep event involving angiogenesis, local invasion, cell migration, dissemination and growth at a secondary site. During malignant transformation, tumour cells have to acquire new functions such as self-sufficiency in growth signals, insensitivity to growth-inhibitory (antigrowth) signals, resistance to apoptosis, adhesive and invasive capacities. Basement membrane provides a main physical barrier to cell migration at different steps of this metastatic process: escape from the primary tumour in carcinomas, intravasation and extravasation during haematogenous dissemination. Invasion of basement membranes involves: (i) the adhesion of tumour cells via cell surface molecules, (ii) secretion of proteolytic enzymes including at least serine proteases and matrix metalloproteases (MMPs) and (iii) cell migration into target tissue in response to specific chemotactic stimuli (;; ).

In addition, the acquisition of invasive properties by some tumour cells is associated with the alteration of several classes of proteins involved in cell–cell and cell–matrix interactions. These affected proteins include at least members of the immunoglobulin and calcium-dependent cadherin families and integrins. The proteolytic event induced by proteases allows the invasion of cancer cells locally and at distance, as well as the release or the activation of growth or angiogenic factors (for review, see;;; ). By using the Boyden chamber assay, we have evaluated different coumarin derivatives as anti-invasive compounds and analysed their inhibitory activity against some serine proteases and MMPs.

In previous works, we have investigated a series of original coumarin derivatives (6-substituted 2-oxo-2 H-1-benzopyran-3-carboxylic acid analogues). Some of them were potent inhibitors of serine proteases such as α-chymotrypsin ( α-CT), human leucocyte elastase (HLE) and thrombin (THR) (,; ).

Based on the invasive assay, among eight coumarin derivatives evaluated, we have selected two compounds, 3-chlorophenyl 6-acetoxymethyl-2-oxo-2 H-1-benzopyran-3-carboxylate (compound 4) and 3-bromophenyl 6-acetoxy-methyl-2-oxo-2 H-1-benzopyran-3-carboxylate (compound 7) , which were further examined for their ability to affect the tumorigenicity in vivo of human fibrosarcoma and human breast adenocarcinoma cells. Their inhibitory potency against different serine proteases and MMPs was also determined. We provide evidence that these two coumarin derivatives display interesting and important in vivo antitumour effect. Coating of culture wells with type IV collagen Plates (24-well) (Falcon, Becton-Dickinson, USA) and 6.5 mm polycarbonate filters (8 μm pore size) of Transwell cell culture chamber inserts (Costar, Netherlands) were coated with 100 μl of type IV collagen purified from human placenta diluted at 200 μg ml −1 in phosphate-buffered saline (PBS). Coats were left to air-dry overnight in a laminar flow hood under UV light. Uniformity of the coating was assessed by Coomassie blue staining. Before use, coated plates and inserts were washed twice with water and incubated for at least 1 h in serum-free DMEM at 37°C.

Invasion assay HT1080 cells exponentially growing in T-75 flasks (Nunc, Costar, Netherlands) were collected by trypsin–ethylenediaminetetraacetic acid treatment, washed with serum-containing medium and allowed to recover from trypsinisation for at least 30 min at 37°C. Cells were then washed twice in serum-free DMEM and diluted in the same medium supplemented with 0.1% bovine serum albumin (BSA, fraction V, Sigma, USA) to a cell density of 6 × 10 5 cells ml −1. The influence of coumarin derivatives (0.1, 1 and 10 μ M) on HT1080 cell invasion and migration was assayed using Transwell cell culture inserts (Costar) and compared to the reference compound GI 129471 (1 μ M), an MMP inhibitor. For chemoinvasion assays, type IV collagen-coated membranes (see above) were used, whereas uncoated membranes were employed for chemotaxis assays. Lower wells of chambers were filled with DMEM supplemented with 20% FCS and 1% BSA (fraction V, ICN) as chemoattractant, in the presence or absence of coumarin derivative (final concentration 0.1, 1 and 10 μ M) or GI 129471 (final concentration 1 μ M). Cells (6 × 10 4) suspended in DMEM supplemented with 0.1% BSA (fraction V, ICN) were seeded in the upper wells. Chambers were subsequently incubated for 48 and 4 h for chemoinvasion and chemotaxis assays, respectively, in a humid atmosphere at 37°C (in order to maintain a chemotactic gradient, media from both lower and upper wells were renewed after 24 h).

After incubation, filters were removed from the chambers, fixed in 4% paraformaldehyde for 15 min (4°C), permeabilised for 10 min in methanol (−20°C) and stained with Giemsa (4%) for 15 min. The upper surface of the filters was scrapped with a cotton swab and cells having reached the lower surface of the filters were visually counted using a light microscope (Vanox AH3, Olympus, Hamburg, Germany) at 20-fold magnification. Each assay was performed in triplicate.

Results were expressed as percentages (±s.d.) of the migration of control cells. Cytotoxicity assay In order to assess the potential influence of coumarin derivatives (0.1, 1 and 10 μ M) on cell metabolism, HT1080 cell suspension (6 × 10 5 cells ml −1) was plated on type IV collagen-coated 96-well plates, 200 μl well −1 (1.2 × 10 5 cells), in the same culture conditions as described above and incubated for 48 h in the presence of different concentrations of the coumarin derivative (medium renewal occurred after 24 h). Media were subsequently harvested and replaced by 100 μl of DMEM supplemented with 10% WST-1 (4-3-(4-iodophenyl)-2-(4-nitrophenyl)-2 H-5-tetrazolio-1,3-benzene disulphonate, Boehringer Mannheim, Germany) and plates were incubated for 2 h at 37°C.

Cellular metabolism was determined by measuring the absorbance of the medium between 420 and 480 nm (with a reference filter of 595 nm) on a microtitre plate reader (Multiscan MS, Labsystems, Finland). At the concentrations tested, the synthetic coumarin derivatives did not significantly affect cellular metabolism (data not shown).

Proliferation and adhesion assays Cells (6.8 × 10 4 for proliferation assay and 1 × 10 5 for adhesion assay) were plated on plastic or type IV collagen or matrigel in 24-well plates (Falcon, Becton Dickinson, USA). At each time points (2 h, 4 h, 7 h 30 min and 23 h for the adhesion assay; days 1, 2, 5 and 7 for proliferation assay), the medium was removed, and the cells were washed with PBS and frozen at −20°C. The DNA content was determined by spectrofluorimetry. For cell adhesion, the DNA content of the initial cell suspension was considered as 100%. Each assay was performed in triplicate. In vivo studies Matrigel (basement membrane proteins) was extracted from the mouse Engelbreth–Holm–Swarm (EHS) tumour as previously described.

Tumoral cells were detached by trypsinisation, harvested by centrifugation, resuspended in 0.25 ml of serum-free medium and mixed with an equal volume of cold matrigel (10 mg ml −1). The suspension (1 × 10 6 cells injection site −1) was injected subcutaneously (s.c.) on both body flanks of 6–8-week-old female athymic nude mice ( n=6; nu/nu Swiss mice from Iffa credo, L'Arbresle, France).

The coumarin derivatives in PBS were injected intraperitoneally (30 mg kg −1) three times per week during the whole assay. Animals received human care, and experiments have been carried out with ethical committee approval. The ethical guidelines that were followed meet the standards required by the UKCCCR guidelines. The tumorigenicity defines the capacity of cells to induce tumour formation. The larger (a) and smaller (b) diameters of the apparent tumour were measured and served for tumour volume calculation according to a × b 2 × 0.4. Injected mice were examined weekly.

Tumours presenting a volume higher than 80 mm 3 and which maintained a progressive growth were taken into account. Results are expressed as the mean (±s.e.m.) of the tumour volumes. Sodium dodecyl sulphate – polyacrylamide gel electrophoresis and zymography The culture medium of human fibrosarcoma HT1080 cells (DMEM) was removed and concentrated 20-fold in a Vivaspin 6 ml concentrator. It was incubated for 30 min at ambient temperature in nonreducing sample buffer and run under Laemmli conditions by loading a volume of 25 μl onto 7.5% polyacrylamide gels containing 0.1% sodium dodecyl sulphate (SDS), 2 mg ml −1 casein and 0.16 μ M human plasminogen. Following electrophoresis, gels were incubated for 1 h in 2.5% (v v −1) Triton X-100 for removal of SDS and then for 1 h at 37°C in 50 m M Tris-HCl, 0.1 M NaCl, pH 7.6. Plasminogen activator (PA) activity was visualised as clear bands of lysis upon staining with 0.1% Coomassie blue R250.

Purified human high-molecular-weight two-chain urokinase plasminogen activator (HMW-uPA) and low-molecular-weight urokinase plasminogen activator (LMW-uPA) were run in each gel as positive controls. An analogous protocol was followed to detect enzymatic activities towards MMP-2 and MMP-9 except that the 7% SDS–PAGE contained 1 mg ml −1 bovine gelatin. The gels were incubated at 37°C for about 18 h in 5 m M Tris, pH 7.6 containing 0.2 M NaCl, 5 m M CaCl 2 and 0.02 (w v −1) Brij35.

Protein staining was performed during 1 h with a 0.25% solution of Coomassie brillant blue R250 in 50% methanol and 7% acetic acid and destaining in a solution of 20% methanol and 50% acetic acid. Purified pro-MMP-2, pro-MMP-9 and MMP-2 were used as positive controls.

MMP-2 was freshly prepared by incubating pro-MMP-2 with 100 μ M p-aminophenylmercuric acetate (APMA, dissolved in 50 m M Tris) for 30 min at 37°C. Some experiments were carried out with the culture medium after activation by 100 μ M APMA (30 min at 37°C). The effects of compounds 4 and 7 (1–100 μ M) incubated in the culture medium were checked for both types of zymography.

Enzymatic studies Bovine α-CT and LMW-uPA (MW=33 000 Da) were purchased from Sigma, HMW-uPA (MW=54 000 Da) from Choay, trypsin (TRY) and THR from Boehringer-Indelheim (Germany), and MMP-2 and MMP-9 from Calbiochem. Human leucocyte elastase and PM were from Elastase Products Co. And Biogenic, respectively. Except for THR, HMW-uPA, MMP-2 and MMP-9, enzyme concentrations were determined by active-site titrations using the appropriate titrant (;; ). Several enzymes were assayed spectrophotometrically at 405 nm using the following p-nitroanilide substrates: succininyl-Ala 2-Pro-Phe- p-nitroanilide (Suc-AAPF- p-NA), benzoyl-Arg- p-nitroanilide (Bz-R- p-NA) and methoxysuccinyl-Ala 2-Pro-Val- p-nitroanilide (MeO-Suc-AAPV- p-NA) for α-CT, TRY and HLE, respectively (obtained from Sigma); or S-2238 (H-D-Ile-Pro-Arg- p-nitroanilide), S-2444 (. At low inhibitor concentrations, the ratio k i/ K I was obtained as k obs/I 0. The enzyme, coumarin derivative and substrate concentrations were: LMW-uPA 0=12 n M, I 0=0.5–10 μ M, S 0=30 μ M; PM 0=28 n M, I 0=2.8–28.3 μ M and S 0=0.14 m M.

In the progress curve method, the substrate competes with the inhibitor and the kinetic parameters are determined as previously described. The enzyme, substrate and coumarin derivative concentrations were: HLE 0=30 n M, S 0=100 μ M and I 0=0.05–70 μ M. The products resulting from the spontaneous hydrolysis of compounds 1–4 and 7 obtained by 18 h incubation at pH 7.5 were also checked for a possible inhibitory effect against the purified enzymes MMP-2 and MMP-9 (50 p M and 1 n M, respectively; S 0=220 μ M), LMW-uPA and HMW-uPA (12 and 15 n M, respectively; S 0=30 μ M) and PM (17 n M, S 0=270 μ M).

Synthetic coumarin derivatives reduce HT1080 fibrosarcoma cells invasion To determine whether coumarin derivatives could reduce the invasive behaviour of tumour cells, we measured the ability of cells treated or not with the coumarin derivative to pass through type IV collagen-coated Transwell cell culture inserts (chemoinvasion assay). Cell invasion in the absence of coumarin derivative was considered as 100% (, Control). The results were compared to those observed with a well-known hydroxamate-MMP inhibitor: GI 129471, used at 1 μ M concentration as reference compound. In such a chemoinvasion assay, this reference compound led to a 60% inhibition of HT1080 cell invasion. Synthetic coumarin derivatives reduce HT1080 fibrosarcoma cells invasion. HT1080 chemoinvasion was evaluated in Transwell cell culture inserts coated with type IV collagen. HT1080 cells (6 × 10 4) were seeded in the absence (control) or presence.

Coumarin derivatives 1–4 reduced HT1080 cell migration in a dose-dependent manner. The unsubstituted derivative 1 (Y=H) reduced the invasion by 50% at 1 μ M and 65% at 10 μ M. The 6-methyl 2 (Y=CH 3) and the 6-acetoxymethyl 4 (Y=CH 2OCOCH 3) coumarin affected the invasion in a similar manner. At 1 μ M, both compounds led to a 50% inhibition, and at 10 μ M, a 70 and 90% inhibition were observed for the methyl 2 and the acetoxymethyl 4 derivatives, respectively. In contrast, cell invasiveness was not markedly affected by addition of the 6-chloromethyl-substituted derivative 3 (Y=CH 2Cl) (only 30% invasion inhibition at 10 μ M).

Thus, these data indicate that an acetoxymethyl group in the 6-position gave rise to better inhibition of cell invasion. We next synthesised the 3-phenyl ( 5) and the 3-halogenophenyl 6-acetoxymethyl-2-oxo-2 H-1-benzopyran-3-carboxylate derivatives 6–8. The different halogen atoms (F, Cl, Br or I) present on the 3′-position gave rise to different effects on cell invasion. Addition of the unsubstituted derivative 5 (X′=H) has a lower inhibitory effect on cell chemoinvasion than that observed with GI 129471. Indeed, when this derivative was used at 1 μ M, a 30% inhibition of cell invasion was observed. At 10 μ M, it gave rise to a similar inhibition (50–60%) as that observed by using the reference compound at 1 μ M. Substitution by a halogen atom improved the inhibitory capacity of coumarin derivatives as compared to the nonsubstituted one.

Addition of chloro (X′=Cl, compound 4) and iodo (X′=I, compound 8) derivatives at 1 μ M led to an inhibition quite similar to that observed with the reference compound. Interestingly, fluoro (X′=F, compound 6) and bromo (X′=Br, compound 7) derivatives appeared more potent, inducing an 80% inhibition at similar concentration. Moreover, chloro (compound 4) and bromo (compound 7) derivatives used at 10 μ M dramatically decreased the cell invasiveness (±90% invasion inhibition). Since these two products (compounds 4 and 7) appeared to be very potent inhibitors of cell invasion, their anti-invasion and anticancer capacities were further studied in vitro and in vivo. We compared the ability of cells treated or not with the two selected coumarin derivatives to invade through type IV collagen-coated filters (chemoinvasion) and through uncoated filters (chemotaxis). Chemoinvasion corresponds to the capacity to invade through an extracellular matrix, while chemotaxis relies on the ability to migrate in response to a chemotactic factor. Addition of the chloro derivative 4 caused a potent and dose-dependent decrease of HT1080 cell chemoinvasion confirming the results reported in.

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In the chemotaxis assay, a similar dose response inhibitory effect was observed (20% inhibition at 0.1 μ M, 50% inhibition at 1 μ M and 80% at 10 μ M). In contrast, cell migration through uncoated filters was not markedly affected by the reference compound at 1 μ M (20–30% chemotaxis inhibition vs 60% inhibition of chemoinvasion). Although the bromo derivative (compound 7) reduced cell invasion and migration in a dose-dependent manner, this compound was a more potent inhibitor of chemoinvasion than chemotaxis.

Its effect on chemotaxis at 1 μ M was similar to that observed with the reference compound. Effect of synthetic coumarin derivatives on HT1080 fibrosarcoma cell invasion. Chemoinvasion and chemotaxis were evaluated in Transwell cell culture inserts coated or not with type IV collagen, respectively. HT1080 cells (6 × 10 4) were seeded. These effects of both compounds could not be ascribed to a modulation of cell adhesion or cell proliferation. Indeed, independent of the presence of coumarin derivatives, 100% of cell adhesion to plastic, type IV collagen or matrigel was reached within 7 h 30 min or 2 h, respectively.

Similarly, cell proliferation was not affected by both compounds (data not shown). Enzymatic studies To further gain insights into the characterisation of the selected coumarin derivatives tested ( 4 and 7), we have evaluated their activity against serine proteases and MMPs released by HT1080 cells. We first performed plasminogen and gelatin zymographic analysis on HT1080 cell conditioned media. Plasminogen activator activity was detected in all samples.

Densitometric analysis of zymograms revealed greater amounts of HMW-uPA than LMW-uPA. Gelatin zymography demonstrated the presence of pro-MMP-2 (66 kDa), pro-MMP-9 (92 kDa), two MMP-2 activated forms (62 and 59 kDa) and a 120 kDa gelatinolytic species. The latter activity was attributed to a complex formed between TIMP-1 and pro-MMP-9. The addition of compounds 4 or 7 to the conditioned culture medium did not abrogate neither the HMW- and LMW-uPA nor the gelatinolytic activities. Matrix metalloprotease production by HT1080 cells analysed by gelatin zymography of the conditioned medium. The 59 and 120 kDa forms were present as traces. Thereafter, we compared the inhibitory potencies of the two selected compounds to that of other coumarin derivatives against purified enzymes.

It was previously demonstrated that the 6-chloromethyl derivative (compound 3) acted as a suicide substrate leading to enzyme irreversible inhibition of α-CT, whereas HLE was transiently inactivated through the formation of a stable acyl-enzyme (,). We found here that LMW-uPA and PM were inactivated by compound 3. No reactivation of inhibited LMW-uPA was observed either spontaneously after 18 h incubation or after treatment with NH 2OH (pH 7.5, 25°C) demonstrating that the observed irreversible process is not because of the formation of a stable acyl-enzyme. Hydroxylamine is known to cleave acyl-enzymes. It was verified that no noticeable denaturation of untreated LMW-uPA occurred during the above experiments.

Presumably, compound 3 inactivates LMW-uPA according to the same mechanism as demonstrated for the inactivation of α-CT (suicide inhibition; ). The closely related dihydrocoumarins (3,4-dihydro-3,4-dibromo-6-bromomethylcoumarin and 3,4-dihydro-3-benzyl-6-chloromethylcoumarin) were demonstrated to act as suicide substrates of LMW- and HMW-uPA with modification of the active site His57 (; ). In sharp contrast, purified LMW-uPA was not inactivated by compounds 1, 2, 4 and 7.

In addition, the activities of purified HMW-uPA, PM, MMP-2 and MMP-9 were not affected by the presence of compounds 4 and 7, or of their hydrolysis products. Surprisingly, the 3-chlorophenyl 6-chloromethyl-2-oxo-2 H-1-benzopyran-3-carboxylate 3, which possesses an inhibitory potency against uPA and PM, did not markedly decrease the cell invasiveness. It is worth noting that the two compounds ( 4 and 7), which markedly decreased the cell invasiveness at 10 μ M (Y=CH 2OCOCH 3; X′=Cl and Br), were unable to act as uPA, PM or MMPs inhibitors. Effect of coumarin derivatives on tumour growth in vivo We next investigated the influence of the two selected compounds 4 and 7 (Y=CH 2OCOCH 3; X′=Cl and Br) on the development of tumours induced by the injection into nude mice of human breast adenocarcinoma and fibrosarcoma cells. Treatment of mice with bromo derivative (compound 7) was found to reduce both the incidence (log Rank test, P. ( A) Incidence of tumours induced by subcutaneous (s.c.) injection of HT1080 cells (percent of animals bearing tumour higher than 80 mm 3). ( B) Growth curves of tumour formed, in nude mice ( n=6), after s.c.

Injection of HT1080 cells (1 ×. The effects of both compounds were also evaluated on the tumorigenicity of mammary MDA-MB231 cells. The MDA-MB231 cell inoculation led to 100% tumours within 2–3 weeks. While the bromo derivative did not affect tumour incidence, it again reduced significantly tumour growth. (ANOVA analysis, P.

DISCUSSION This paper describes the evaluation of the anti-invasive properties of recently developed synthetic coumarin derivatives. The starting point of this work was the observation that 2-oxo-2 H-1-benzopyran-3-carboxylic acid derivatives act as potent inhibitors of diverse serine proteases such as α-CT, HLE and THR (,; ).

Owing to possible implication of serine proteases such as uPA and PM in the metastatic process (; ), a selection of previously described coumarins as well as new synthetic derivatives was therefore examined in vitro and in vivo on different experimental models. Thus, in order to highlight possible anticancer properties linked to their expected profile as protease inhibitors, the inhibitory capacity of the different compounds was determined in vitro on several serine proteases and MMPs (MMP-2 and MMP-9). Moreover, chemoinvasion and chemotaxis in vitro assays as well as in vivo tumorigenic assays were conducted on nude mice, inoculated with HT1080 or MDA-MB231 cells. Using noncytotoxic concentrations of compounds, our studies illustrated that some coumarin derivatives markedly inhibited the HT1080 cell invasiveness.

The inhibition capacity varied according to the substituent present in the 6-position of the coumarin, and according to the nature of the halogen atom in the 3-position of the phenyl ring. In general, substitution by a halogen atom (particularly, a chlorine or a bromine atom) in the ‘meta’ position of the phenyl ring relative to the ester oxygen atom of 2-oxo-2 H-1-benzopyran-3-carboxylate led to a better anti-invasive effect than that observed in the absence of any substituent. In the same way, an acetoxymethyl or a methyl group in the 6-position conferred an improved effect. It is worth noting that 3-chlorophenyl 6-acetoxymethyl-2-oxo-2 H-1-benzopyran-3-carboxylate (compound 4) has almost the same potent inhibitory activity on cellular invasion (chemoinvasion) and on cell migration (chemotaxis). In sharp contrast, the corresponding 3-bromo derivative (compound 7) led to a low inhibitory effect on chemotaxis, similar to that observed with GI 129471 used as reference compound (30% inhibition at 1 μ M). Interestingly, the bromo derivative was more potent in the chemoinvasion assay than the reference compound and the chloro-substituted analogue. These data emphasise the interest of the bromo derivative as an anti-invasive inhibitor.

On the contrary, the chloro derivative which affected both chemoinvasion and chemotaxis is likely to modulate cell mobility in a manner independent of a protease inhibition. In accordance with these in vitro observations, our in vivo study in two experimental models demonstrates that only the bromo derivative markedly affected tumour growth. It reduced both the incidence and the growth of tumour induced by HT1080 cell injection. Furthermore, it affected the growth of more aggressive breast adenocarcinoma MDA-MB231 derived tumours. The two selected coumarin derivatives 4 and 7 were not found to act as an uPA, PM, MMP-2 or MMP-9 inhibitor. Moreover, the coumarin derivative 3, which was found to be a strong inactivator of α-CT and THR and which possessed an inhibitory potency towards uPA, did not markedly decrease cell invasiveness.

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Thus, the observed activity of the coumarin derivatives on HT1080 cell invasion could not be ascribed to their inhibitory activity against the enzymes tested. Both compounds were found to be unable to inhibit the angiogenesis evaluated in the aortic ring assay (data not shown) according to the procedure previously described (; ). Consequently, further experimental investigations are required to determine the exact mechanism of action of the bromo coumarin derivative and its biological target. For example, the recently described MT-SPS which are membrane-bound serine proteases implicated in tumorigenesis are possible targets for protease inhibitors like coumarins (; ). In the light of our in vivo and in vitro results, the bromo coumarin derivative appears to be very promising as potential antitumoral agent. To the best of our knowledge, this study provides for the first time evidence that a coumarin derivative displays marked anti-invasive properties in vitro and antitumour activities in vivo. The disappointment of MMP inhibitors in clinical trials as a result of the expression of a high toxicity emphasises the importance of a better elucidation of the mechanism of inhibition action in vitro and in vivo.

Although coumarin derivatives might constitute an alternative to MMP inhibitors as anticancer agents, further biological investigations are required before any clinical trial.