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Popular property highlights
Hyatt House Seattle/Bellevue
Property Amenities
Property Amenities
Hyatt House Seattle/Bellevue features an indoor pool and a fitness center. The aparthotel offers a restaurant and a snack bar/deli. A bar/lounge is on site where guests can unwind with a drink. Guests can enjoy a complimentary breakfast as well as a complimentary manager's reception (may not be available daily). High-speed wireless Internet access is complimentary.
A business center is on site at this 3-star property. This business-friendly aparthotel also offers barbecue grills, multilingual staff, and coffee/tea in a common area. Onsite self parking is complimentary.
Hyatt House Seattle/Bellevue is a smoke-free property.
- 24-hour front desk
- Bar/lounge
- Barbecue grill(s)
- Business center
- Coffee/tea in common areas
- Free newspapers in lobby
- Free breakfast
- Concierge services
- Dry cleaning/laundry service
- Elevator/lift
- Express check-out
- Fireplace in lobby
- Fitness facilities
- Free WiFi
- Laundry facilities
- Multilingual staff
- Number of floors - 5
- Total number of rooms - 160
- Luggage storage
- Restaurant
- Safe-deposit box at front desk
- Snack bar/deli
- Free reception
- Free self parking
- Smoke-free property
- Indoor pool
- Television in common areas
Family Friendly Amenities
- Indoor pool
- Free Wi-Fi
- Snack bar/deli
- Laundry facilities
- Cribs/infant beds (complimentary)
Available in all rooms: Free WiFi , Free wired high-speed Internet
Available in some public areas: Free WiFi
Free self parking
Room Amenities
Hyatt House Seattle/Bellevue offers 160 air-conditioned accommodations with coffee/tea makers and complimentary weekday newspapers. Pillowtop beds feature premium bedding. Flat-screen televisions come with premium satellite channels. Bathrooms include designer toiletries, complimentary toiletries, and hair dryers.
This Bellevue aparthotel provides complimentary wired and wireless high-speed Internet access. Business-friendly amenities include desks and phones. Housekeeping is provided daily.
- Air conditioning
- Phone
- Free toiletries
- In-room climate control (air conditioning)
- Coffee/tea maker
- Designer toiletries
- Desk
- Free weekday newspaper
- Hair dryer
- Iron/ironing board
- Satellite TV service
- Premium bedding
- Premium TV channels
- Daily housekeeping
- Free cribs/infant beds
- Flat-panel TV
- Free WiFi
- Free wired high-speed Internet
- Pillowtop mattress
Where to Eat
Guests are offered a complimentary hot/cold buffet breakfast. The aparthotel offers a complimentary manager's reception on select days.
Hyatt House Seattle/Bellevue has a restaurant on site.
Nearby Things to Do
Recreational amenities at the aparthotel include an indoor pool and a fitness center.
The recreational activities listed below are available either on site or nearby; fees may apply.
- Downhill skiing nearby
- Cross-country skiing nearby
- Snowmobiling nearby
- Scuba diving nearby
- Snorkeling nearby
- Personal motorized watercraft on site
- Hiking/biking trails nearby
- Mountain biking nearby
If you have requests for specific accessibility needs, please note them at check-out when you book your room.
- Accessible bathroom
- Roll-in shower
- In-room accessibility
Property Policies
Check-in time ends at midnight
Check-in time starts at 3 PM
Minimum check-in age is 21
Check-out time is noon
Payment types
You need to know
Extra-person charges may apply and vary depending on property policy.
Government-issued photo identification and a credit card are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
Service animals exempt from fees/restrictions
No rollaway/extra beds available
Property Name
- HYATT house Seattle/Bellevue
- HYATT house Seattle/Bellevue Aparthotel
- HYATT house Seattle/Bellevue Aparthotel Bellevue
- HYATT house Seattle/Bellevue Bellevue


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When players start building a reputation, more eyes start focusing on them. That can be said about the Minnesota Vikings secondary, which is coming off two of the most dominating shut-down performances against opposing wide receivers that the Vikings have put together in years.
Can they make it three in a row?
That's what makes the head-to-head assignments between Vikings cornerbacks Xavier Rhodes, Terence Newman, Trae Waynes and Captain Munnerlyn going up against Houston's DeAndre Hopkins and Will Fuller V this week's key matchup.
The Vikings have due credit to give to all three levels of the defense for what the team has accomplished to date this season, but, by and large, there were no real big surprises at most of the positions.
With all things equal without further injury, the Vikings knew they had a dominant defensive tackle in Linval Joseph, a dominant defensive end in Everson Griffen and quality depth all along the line. They knew they had two of the better young linebackers in the league in Anthony Barr and Eric Kendricks. They knew they had an All-Pro safety in Harrison Smith.
They also knew they had talent and an investment in the cornerback spot. They used not one, but two first-round picks on cornerbacks (Rhodes and Waynes) and spent the first two years of the Mike Zimmer era adding veteran cornerback Zimmer was convinced would thrive in his system (Munnerlyn and Newman).
They knew they had the component parts in place, but how would the four of them function as a unit to propel the Vikings from a very good team to an elite team?
Rhodes missed the first two games of the season with a knee injury that struck during warmups prior to the regular season opener. Just as the Vikings have dealt with offensive adversity, losing their top cornerback nixed the original plan for the secondary.
In his absence, the key component of the cornerback Fab Four – Waynes – needed to step up. As a rookie, Waynes was used sparingly. He was full-time and prime-time the first two games. He over-performed expectations, while Newman and Munnerlyn did full-time duty.
In the two games since Rhodes has been back, he has been largely been assigned to the top receiver of an opponent. If you look at the stat sheet, it doesn't look that impressive, yet it was.
Kelvin Benjamin of the Carolina Panthers was Rhodes' assignment in Week 3. Rhodes didn't have a tackle. He didn't need one. He blanketed Benjamin and Cam Newton only targeted him once and that pass was incomplete.
What the Vikings did last week against the Giants was more impressive. New York came into U.S. Bank Stadium with three legitimate receiving threats in Odell Beckham Jr., Sterling Shepard and Victor Cruz.
It wasn't simply going to be Rhodes who needed to do his job. It was the combination of Newman, Waynes and Munnerlyn to take care of their job as well. The Vikings mixed and matched the three outside guys and Munnerlyn was replaced by Chad Greenway for only two plays in the defensive formations.
Eli Manning was willing to throw on every down and the Vikings responded defensively. In the process, Beckham had a career-low 23 yards receiving and made headlines for all the wrong reason.
The Texans will be just the opposite. Houston wants to run, which might have the base defense on the field more often than it has been since the Tennessee game (although the Texans also like to use three and four receivers).
Only the Cowboys' Ezekiel Elliott has more rushing attempts (94) than Houston's Lamar Miller (93). Since the days of Arian Foster being a young up-and-comer, Houston has run the ball like few others. Miller has never been a bell cow running back. In Miami, he was a featured back, but only averaged about 13 carries a game.
In Houston, that's different. In four games, he has 93 carries (weekly totals of 28, 25, 21, 19 in that span) and the expectation is that he will be carrying a heavy load against the Vikings. As a result, there could be one less cornerback on the field Sunday for many plays – most likely Munnerlyn taking the biggest hit.
More times than not, the Vikings corners will be singled up on Hopkins and Fuller. Hopkins is an elite receiver who has been the passing centerpiece of the Texans' offense since he arrived. He ran out Andre Johnson, no small feat for a legacy player like him. Hopkins is a playmaker who thrived despite a carousel of quarterbacks, each less talented than the one before.
By Hopkins standards, he isn't having the type of start he has reached historically. He has 17 catches for 227 yards through four games. But, he is coming off his last game – vs. Tennessee – where he caught just one pass for four yards. He is angry, much like Beckham was angry last week. He wants the ball and Brock Osweiler will have to be convinced to make good on giving Hopkins the chance to shine.
Fuller has been the most dominant wide receiver in the rookie class of 2016. Houston flip-flopped picks with Washington in front of the Vikings to make sure they landed Fuller. They invested more than the rest to acquire a receiver they clearly coveted.
So far, so good.
Through four games, Fuller leads the team in receptions (19), yards (323) and average per catch (17.0 yards). Teams are now game-planning for Fuller.
If Rhodes is assigned Hopkins, the combo platter of Newman and Waynes will be assigned Fuller. They may mix things up occasionally with Braxton Miller in the slot, but that will be the standard face-off when the ball is snapped.
The Vikings are playing dominant defensive football and, it can be argued, the two games in which they've had all four of their primary cornerbacks together are the ones in which they have thrived most.
Given how often the Vikings defense will be forced to stop the run – Houston doesn't give up on the run often, even when they got their doors blown off by New England – the threat of the big play over the top will be what the Houston offense is hanging its hat on, making that chess game this week's key matchup.null


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Layoffs Hit Autodesk Employees
There are 6 comments on the WorldCAD Access story from Apr 17, 2008, titled Layoffs Hit Autodesk Employees. In it, WorldCAD Access reports that:
The Marin Independent Journal reports Autodesk is cutting jobs for the reason of efficiency: The San Rafael-based computer-aided design software company made the cuts to employees in its Platform Solutions and ... via WorldCAD Access
Join the discussion below, or Read more at WorldCAD Access.
Saskatoon, Canada
#1 Apr 17, 2008
Maybe they should re-assign them to programming positions to get AutoCAD MAP 3D to do actual GIS work ... especially the simple stuff like TRANSPARENCY!! The current programmers can't seem to get Map 3D to do what other GIS packages have been doing for years. I'm going back to ESRI, maybe the laid off employees should too.
I wonder if Carl Bass would take a pay cut to keep some staff ... especially since he is such a great leader (CAD Society Leadership Award)?!
#2 Apr 28, 2008
This is all part of our plan of record to move R&D to our china development office. We will be able to elminate the fat and expensive staff in Marin, and hire excellent china's programmers, and enter that market with Strength.
Chien Ming
#3 Jan 15, 2009
I guess that really sucks for the Marin staff eh?
Ottawa, Canada
#4 Jan 18, 2009
I take it that you mean a larger one than the 10 percent cut he announced for himself and his senior VPs?
Kingston, Jamaica
#5 Jan 16, 2010
You have to understand that ESRI doesn't lay off people or fire them. They squeeze them out when they see fit to reduce staff or get rid of an employee that might be making too much money or may have cost the company money while on disability. Trust me, I worked there as a software developer for 22 years. Core programmer, one of the first five programmers. Once I was expendable after my heart surgery, they squeezed me out. I've seen it over dozens of years. Don't let the false store front confuse you. So many of my former employees are miserable and just barely holding on to their jobs. Dangermond is shielded from the daily crap by management for fear of them loosing their own jobs. Its really sad because back in the 80's and 90's it used to be the coolest place to work. Most of us would say around the coffee pot that we would work here for free as long as we had a roof over our head and food on the table. Am I bitter? Sure a bit, but after being squeezed out by Don Barry (third in command) for supposedly costing the company US$70M while being on disability for a bad heart from going to the dentist, I lost all respect for the company and Dangermond. The fact that after 22 years he didn't even call me or have someone visit. All I got was a phone call that Chuck from HR was coming to my home to take all my computer equipment away after I had heart surgery. Trust me, ESRI is a company after one thing. To take your money, blind you with false promises. Don't get me wrong. "most" of the worker bees at the company are awesome. They are devoted, smart, and work their asses off. But management is blind. Including people like Keith Ryden who runs his department just like Julius Caesar.
#6 Mar 29, 2010
Autodesk continues to orchestrate layoffs. The difference is that now the RIFs are just smaller to keep them off the national radar. They just laid off a bunch of documentation folks last month.
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Residents are advised that on Saturday May 27 we will be using a mechanical sweeping device to clean the remaining sand and debris from sidewalks.Sweeping will commence at 7:00 AM and will take 3-4 hours to complete. Since this is a dry sweeping method there may be dust generated by the sweeping. We highly recommend that residents who live on streets with sidewalks keep all windows on the front of their houses closed until the sweeper has passed. We ask that residents not park vehicles in a manner that blocks any portion of the sidewalk, or on the street adjacent to the sidewalk as the sweeper will be directing the sand and debris toward the street.We will not be able to sweep sidewalks in areas where vehicles are parked on the street due to the risk of damaging vehicles.
The Town of Massey Drive will once again be hiring students for summer employment under the Canada Summer Jobs program. Any students interested in working for the Town this summer are encouraged to drop off a resume at the Town Office. Resumes will be accepted until Wednesday June 7, 2017 and may be dropped off at the Town Office between 8:00 AM and 4:00 PM Monday to Friday or sent via email to firstname.lastname@example.com.
Residents of the Town of Massey Drive are advised that the town's Covered Garbage Regulations are now once again in effect. We would like to thank you for your co-operation over the winter months.
Effective immediately all garbage placed at curbside for pick up must be covered or placed in acceptable covered containers.
Acceptable covering material would be:
-Nylon or polyester netting with mesh not larger than 25mm x 25mm.
-A blanket or tarp that provides adequate coverage, does not blow off, and is easily removable by the garbage collector.
All containers or garbage cans must be water tight and be equipped with handles and water tight covers.
Click on any Town Hall category below.
Town Council
Council Minutes
Town Plan
Budget & Taxes
Forms & Documents


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THE One Show's Dan Snow told a jury yesterday how he helped cops nick a riot
thug — by flattening the fleeing looter.
Dan, 33 — the BBC programme's History Hunter — sent the lout sprawling into a
gutter during last summer's mayhem.
He then pinned him down outside his flat in Notting Hill, west London.
Brave Dan said in a statement read out in court: "I felt I could do something.
He ran towards me and I barged my right shoulder into him.
"He fell into the gutter. I was then able to pin him down with my knees. He
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Cops arrived seconds later. Telly historian Dan — whose broadcaster dad Peter
Snow is famed for his election "swingometer" — went outside after hearing a
Two mopeds were on fire, a gang was smashing car windows — and a young Asian
had blood "streaming down his head" as his shop was targeted. The looter he
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Cognitive Vulnerability Can Be 'Contagious' And Predict Episodes Of Depression
The research, from psychological scientists Gerald Haeffel and Jennifer Hames of the University of Notre Dame, is published in Clinical Psychological Science, a journal of the Association for Psychological Science.
Studies show that people who respond negatively to stressful life events, interpreting the events as the result of factors they can't change and as a reflection of their own deficiency, are more vulnerable to depression. This "cognitive vulnerability" is such a potent risk factor for depression that it can be used to predict which individuals are likely to experience a depressive episode in the future, even if they've never had a depressive episode before.
Individual differences in this cognitive vulnerability seem to solidify in early adolescence and remain stable throughout adulthood, but Haeffel and Hames predicted that it might still be malleable under certain circumstances.
The researchers hypothesized that cognitive vulnerability might be "contagious" during major life transitions, when our social environments are in flux. They tested their hypothesis using data from 103 randomly assigned roommate pairs, all of whom had just started college as freshmen.
Within one month of arriving on campus, the roommates completed an online questionnaire that included measures of cognitive vulnerability and depressive symptoms. They completed the same measures again 3 months and 6 months later; they also completed a measure of stressful life events at the two time points.
The results revealed that freshmen who were randomly assigned to a roommate with high levels of cognitive vulnerability were likely to "catch" their roommate's cognitive style and develop higher levels of cognitive vulnerability; those assigned to roommates who had low initial levels of cognitive vulnerability experienced decreases in their own levels. The contagion effect was evident at both the 3-month and 6-month assessments.
Most importantly, changes in cognitive vulnerability affected risk for future depressive symptoms: Students who showed an increase in cognitive vulnerability in the first 3 months of college had nearly twice the level of depressive symptoms at 6 months than those who didn't show such an increase.
The findings provide striking evidence for the contagion effect, confirming the researchers' initial hypothesis.
Based on these findings, Haeffel and Hames suggest that the contagion effect might be harnessed to help treat symptoms of depression:
"Our findings suggest that it may be possible to use an individual's social environment as part of the intervention process, either as a supplement to existing cognitive interventions or possibly as a stand-alone intervention," they write. "Surrounding a person with others who exhibit an adaptive cognitive style should help to facilitate cognitive change in therapy."
According to the researchers, the results of this study indicate that it may be time to reconsider how we think about cognitive vulnerability.
"Our study demonstrates that cognitive vulnerability has the potential to wax and wane over time depending on the social context," say Haeffel and Hames. "This means that cognitive vulnerability should be thought of as plastic rather than immutable."
Association for Psychological Science
Source: EurekAlert!, the online, global news service operated by AAAS, the science society
Visit our Depression category page for the latest news on this subject, or sign up to our newsletter to receive the latest updates on Depression.
Please use one of the following formats to cite this article in your essay, paper or report:
Association for Psychological Science. "Cognitive Vulnerability Can Be 'Contagious' And Predict Episodes Of Depression." Medical News Today. MediLexicon, Intl., 23 Apr. 2013. Web.
29 May. 2017. <http://www.medicalnewstoday.com/releases/259380.php>
Association for Psychological Science. (2013, April 23). "Cognitive Vulnerability Can Be 'Contagious' And Predict Episodes Of Depression." Medical News Today. Retrieved from
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Casares Tourism & Travel Guide
7 Days
18.9k Views
The Quaint Places Of Spain
I suppose I should start with a warning: If you are not a devotee of European history, stick to the 3 C Rule: More than 3 castles and/or cathedrals in one trip and every one starts to look the same....
View more comments...
Top Places To Visit 12 Spots
Church of La Encarnacion
La Fuente, 96, Casares
Casares Cemetery
Casares Castle, Casares
Places To Stay 29 Places
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Located on the A377 less than 15 minutes from Manilva, Casares is named after Julius Caesar, who used the sulphur baths nearby. Casares is also the birthplace of Blas Infante Perez de Vargas, a labor lawyer, writer and important figure in the Spanish Civil war who was executed by Franco in 1935. Casares is the most photographed village in Spain. It offers a 12th century castle, stunning views to the sea, and classic, narrow streets that are filled with bars and cafes. If you visit Casares, stop at the Hedionda Roman Baths (free) for whatever ails you. For some exercise, hike from the base of the city to the museum at the top at sunset.
How To Reach
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29 Hotels found


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Etiquette is when you are invited to a wedding, you buy a gift for the happy couple. Usually this is something that can be used in the home as they start their new home together. Even though the majority of couples these days have lived together before wedlock, the tradition still stands.
As you can imagine, when heading to a wedding of a friend or family member, all the other guests are doing exactly what you re doing and looking for that one perfect gift. Some couples put together a registry of what they need at a specific shop, but if you don't have the budget to buy them the crystal glasses they want or the gold embossed photo frame, then you are going to have to think outside the box and give them something that no one else is going to give to them.
The first thing to remember is the majority of gifts you can buy for the bride and groom are for the kitchen, but rather than giving them something dull and boring, you can focus on providing them with engraved, personalised and unique gifts that they will want to keep, use and enjoy for years to come.


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Item Discontinued
This is a picture of my husband and his older sister when they were kids. They live in different countries now and miss each other so much.
I am still having trouble to take a proper image of the LO, still learning, lol. Recently I started to go through my stash of scraps, I tried to use some of them with this LO, except for the background page the rest is all my leftovers. I also used some mists, I've got to stop misting, just love it!
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- Let the mixture cool to a warm temperature.
- Test the warmth by testing it on your hands, never on your face.
- Once it is ready, apply it on the nose with a help of a brush. Leave it on for 10 minutes and then gently peel off the product from your nose.
- Wash your face. MORE: Turmeric Face Masks for clear skin.
2DIY Deep Cleaning Peel off mask:
- Egg white- 1 egg
- Lime juice- 1 spoon
- Tissue paper
- Brush
- Take a bowl and put the egg white as well as the lime juice into it and whisk it well. Now apply this mixture on the nose and wherever you have blackhead issue on the skin. On top of one coat of egg white place a tissue paper and press it gently.
- Then apply another coat of egg white mixture on the tissue.
- Leave it on until it dries off.
- Then gently peel off the tissue paper. The blackheads will get eliminated with this simple and easy method.
3DIY pore cleansing with honey:
- Honey- 1 tablespoon
- Raw milk- 1/2 tablespoon
- lemon juice – 1/2 tablespoon
- Take a bowl and add raw milk as well as honey and lemon juice into it and place it in the microwave until it becomes a thick as well as a sticky mixture.
- Apply the slightly warm mixture on your face using a brush or a cotton pad and after 15 minutes, rinse it off. Best ways to use honey for skin and hair.
4Clay mask for blackhead removal:
Clay is a natural skin purifier and it also helps in eliminating the blackheads easily. Loose clay is easily accessible from any beauty store. Loose clay not only removes the blackheads from the skin but they also minimize the pore size. This is the reason behind clay mask being popular in every salon and spas.
- Put the clay powder in a bowl and mix it with little apple cider vinegar to make a paste. Then with the help of a brush slather it on the face or on the area affected with blackhead.
- Apply a thin layer and leave it to dry for around 10 minutes. As it dries you will feel your skin tightening.
- Once it dries wash off the mask with water.


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I am a basketball player which for now is the most important thing to me at least while I have cartilage left in my knees :) I am also kind of a nerd though and am looking to get a degree in mechanical engineering with which I plan to go into the automotive feild! (After getting a master's of course) i am also nerdy enough to enjoy things like comic con and video games, definitely not the nerdiest person I know but also far from the lest nerdy. I enjoy other sports and out door activities as well. I also tend to be a very busy person (so please if it takes me a while to respond just bare with me :)) but I love to have fun, whether that's going out or staying in.
And though I've already stated some of these I really enjoy the following
Working out
Video games
Fixing cars
Hanging out
I like a lot of different things :)
my team
my family
My intelligence
Also I would love either some eye candy or an intellectual conversation maybe even both ;)
And remember as with any one just because one person thinks you are attractive, interesting, uninteresting or unattractive doesn't mean everyone sees it that way :)


text:
Corgi was founded in 1892 by Carmarthenshire draper Rhys Jones who, having purchased some second-hand knitting machines, began to produce thigh-high woollen stockings for the local colliers. He then began to produce a range of finer quality socks, ideal for men to wear to chapel on Sundays. From the beginning, Rhys paid very close attention to detail and quality and as the reputation of his products grew, demand spread across the whole of South Wales and beyond.
Donations may only apply to the sale price and not include VAT, delivery and other such costs. Please do not use voucher codes from other websites as this may affect your donation.
Unfortunately there are currently no offers available for this retailer.


text:
- 1[usually passive] repulse somebody to make someone feel disgust or strong dislike synonym repel I was repulsed by the horrible smell. synonyms at shock
- 2repulse somebody/something to fight someone who is attacking you and drive them away synonym repel to repulse an attack/invasion/offensive
- 3repulse somebody/something to refuse to accept someone's help, attempts to be friendly, etc. synonym reject Each time I tried to help I was repulsed. She repulsed his advances. Thesaurusshockappall horrify disgust sicken repulseThese words all mean to surprise and upset someone very much.shock [often passive] to surprise someone, usually in a way that upsets them:We were all shocked at the news of his death.appall to shock and upset someone very much:I was appalled at the way she spoke to her mother.horrify to make someone feel extremely shocked, upset, or frightened:The whole country was horrified by the kidnapping.disgust to make someone feel shocked and almost ill because something is so unpleasant:The level of violence in the movie really disgusted me.sicken to make someone feel very shocked, angry, and almost ill because something is so unpleasant:I was sickened by the lack of concern for the child's welfare.repulse [often passive] (somewhat formal) to make someone feel disgust or strong dislike:I was repulsed by the smell of liquor on his breath.Patterns shocked/appalled/horrified/disgusted/repulsed at somebody/something to shock/appall/horrify/disgust somebody that… to shock/appall/horrify/disgust/sicken somebody to think/see/hear… somebody's behavior shocks/appalls somebody violence/an idea shocks/appalls/horrifies/disgusts somebody
NAmE//rɪˈpʌls//(formal)Verb Forms present simple I / you / we / they repulse
he / she / it repulses
past simple repulsed
-ing form repulsing
Check pronunciation: repulse


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HB Racing Gear Differential Case (40T)
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This is a replacement Hot Bodies Pro5 Gear Differential Case. Package includes diff cup and 40 tooth diff gear.
This product was added to our catalog on July 3, 2015.


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Posted August 27, 2014 by JamesTown in ThatsEnuff
New Artist Spotlight: Better Taste Bureau [Audio]
Had the pleasure to interview rising indie group Better Taste Bureau hailing from Salt Lake City, Utah. The trio consists of Two rappers, Hurris and Gig and producer Mason Brewer. They talk about their debut project "Outliers" a 12 track full-length album and winning City Weekly's Rappers of the Year and U92?s (92.5 FM in SLC) first Homegrown Hip Hop Champions. Stream the interview below:


text:
The Top 25 Android Games
February 17, 2012 - Android gaming is finally here in full force. IGN has whittled down a list of hundreds of great games into 25 definitive must-own titles.
IGN Does Android
July 15, 2010 - Today, IGN launches coverage of Android. To kick things off, we review a host of games, pick out some winning apps, and review of the HTC Evo 4G handset.
Drop7 Android Review
July 15, 2010 - Do you like numbers? And not a in casual way, either. I mean, you covet numbers in a way that would have gotten you stoned in the public square 2,000 years ago. If so, Drop7 is your next Android download.


text: 4/06/09 4/06/09 POPSUGAR Celebrity POPSUGAR Celebrity Gwen and Her Boys at The Beach April 6, 2009 by Celebrity 0 Shares Chat with us on Facebook Messenger. Learn what's trending across POPSUGAR. View on One Page Photo 1 of 16 ADVERTISEMENT () Share This Link Copy ← Use Arrows Keys → 1 Gwen and Her Boys at The Beach Previous Next Share this post


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: Feature
Monday, 13 August 2012
We will never let imperialist powers destroy Syria say Syrian youth in London
A section of the demonstration in support of President Assad and the Syrian Army outside the countrys embassy in London on Saturday
Were here, the Syrian community, at this protest organised by Syrian Youth in Britain, Danny Makki, one of over 60 mainly young people outside the Syrian embassy in London, told News Line on Saturday.
Danny continued: We are here in solidarity with the Syrian people in rejection of new unprecedented EU sanctions, persistent Free Syria Army terrorism and to demand a peaceful solution to our countrys crisis.
We want to show the world and especially the Syrian people that we have a voice and that we will never let the imperialist powers destroy Syria in the name of human rights.
We Syrians believe that the West are supporting Islamist and Jihadi terrorist groups in Syria, and is imposing reprehensible sanctions on an already beleagured people.
My message to the people of Britain, including workers, is to confront the hypocrisy of Western foreign policy and this conspicuous attempt to destabilise a sovereign country.
Syria is paying the price of confronting Western hegemony and supporting national resistance movements in the region.
The West only promotes democracy when it is in their interests.
This is manifested in their unequivocal support for despotic regimes in Qatar and Saudi Arabia, who send their vision of democracy to Syria through suicide bombs and jihadi fighters.
Rizk Rizk added: Weve created a group of youth called Syrian Youth in Britain.
This is the second time we are organising the demonstration outside our embassy here in London.
We are eleven young people, all under 26, who started the group.
We noticed there are so many Syrians living in Britain who wanted to support President Assad and our government and to refuse the armed gangs who call themselves the Free Syrian Army, but to call for dialogue.
Most of the armed gangs are not Syrians.
A British journalist who was kidnapped a few weeks ago said the jihadists who held him were not Syrians none of them were and ten to fifteen were British.
The British government has been supporting the opposition regardless of the fact that most Syrians still support their government and the president.
This is also regardless of the fact that the opposition is armed, targeting civilians and inciting sectarianism.
The government are also misinforming the British people of the reality of the conflict through the media, including the BBC.
Just yesterday, when William Hague decided to give £5m to the opposition, he said it was going to be non-lethal support.
But the oligarchic monarchies in the Arab Gulf are providing weapons to the jihadists in Syria.
So when the British government provides non-lethal support, they make the lethal support of the Arab monarchies more effective and lethal.
This is by providing them with technology and satellite phones. By this, Hague is misleading the British people.
Were here to support the Syrian Army, which is the protector of the Syrian people and the guarantor to the country of Syria.
We are sure the government and the army are going to win.
That is why we are not afraid of demonstrating and showing the truth, because we are the Syrian people.
I heard this morning that the terrorists have been cleared out of Salahddin district in Aleppo.
Journalist graduate Shoruk Khaddour, 23, said: Im here showing support for my country, for the Army and, most importantly, for my people who after 17 months have managed to resist the conspiracy against Syria and fight off terrorism.
Im showing support for what the Army are doing in Aleppo, for what they did in Damascus and for risking their lives to keep us safe.
It is obvious that from the very beginning it has been a Western-backed conspiracy, that includes Saudi Arabia, Qatar, Turkey as well.
Turkish prime minister Erdogan recently said that Iran and the Syrian president are equivalent to the anti-Christ.
It is obvious they are trying to stir sectarian tension.
They play the religion card because they know there are fighters coming from everywhere who think they are fighting a holy war.
A lot of them are just mercenaries.
But the Syrian Army is doing a really good job.
They managed to clear up Damascus and are now doing the same in Aleppo.
A lot of people who say they are leftist here in the UK are giving in to imperialism, even though they say they are anti-imperialism.
We are saying dont fall for the propaganda.
British workers and youth should say no to intervention and let Syrians take control of their country and their future.
Syrians have said no intervention and no to terrorism, so should the British people.
Allaa, 21, also a graduate student, added: Were here to support Syria, specifically to support the people of Aleppo and to reject intervention.
We stand by our president, we stand by the truth.
We support the reforms and the road to democracy that the president outlined.
The West ignores the fact that in 2014 there are going to be presidential elections.
Weve already had local elections and in 2010, the president said in an interview with Dutch TV that democracy is not the means to an end, it is the end.
He said we are working towards that but it doesnt happen overnight.
He was already talking about democracy before the Arab Spring.
I would say to young people in Britain dont rely on one news source, you have to be open-minded.
The British government does have an agenda. They are looking out for their interests, for the Saudi rulers and the Israelis interests.
Student Intisar Abdul Nabi, 22, said: Im here to support the president of Syria.
He is defending our country against Israel and America who want to take over the whole of the Middle East. They are after the oil.
I dont believe the UK foreign secretary William Hague when he said he is giving £5m to the opposition in non-lethal support.
What he is supplying will help kill people.
British workers and young people should stand up against their government.
Everyone is suffering here, they cant get job, everything is going up and they are cutting benefits as well.
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£46,000 Guide price
RICS HOME REPORT - £55,000. TENANTED INVESTMENT PROPERTY - Immaculate and spacious second floor traditional 1 BEDROOM FLAT with wonderful views over Caldwell Bay &beyond to the Rosneth peninsula. Generous room sizes internally with well maintained communal stairwell and private rear gardens. Ample on street parking to the front of the building - Super Flat + location. Accommodation comprises: Reception hallway, lovely bay windowed lounge with front aspects and open views, modern fitted dining sized kitchen with appliances, double bedroom and modern fitted bathroom. Great storage throughout - Rental Potential up to £6,000 per annum - Super location - Excellent building - BIG FLAT. Great location near the town centre amenities, train station and ferry terminal. Internally in excellent order having been recently redecorated and with modern kitchen and bathroom. Original cornicing. Gas central heating throughout. SALES EVIDENCE: 117 Shore Street, Flat 2-3, Gourock, PA19 (SOLD) - £58,500. Sales: 78 Shore Street, Gourock, PA19 1RD (SOLD) for £66,500. PRICED FOR FAST SALE. Overlooking Firth of Clyde near Ferry Terminal &Gourock train station ** Gourock has stunning River Clyde views with neighbouring Greenock less than 10 minutes, Glasgow Airport about 30 minutes with Glasgow City Centre 40 minutes. The town of Gourock has established local shopping and high street banking with more extensive supermarket facilities available in adjoining Greenock. The area has local schooling at both primary and secondary levels &many recreational amenities including several parks, a gym and several golf courses
This house description is based upon information supplied by the owner, or on behalf of the owner. These property particulars are produced in good faith and do not constitute or form part of any contract. s1homes do not take any responsibility for the accuracy of the information contained in this document.
If a property value displays '0', don't worry it just means no properties have been sold in that area during that time.
Sold house price data is supplied to us by the Registers of Scotland, a government department responsible for compiling and maintaining property registers, every month. There can be a delay of up to 3 months from when a property is sold to being recorded with Registers of Scotland. We provide house price data for your information only, we do not accept any liability for any errors. If you have noticed any incorrect information, please contact Registers of Scotland.


text: Assuming I get caught unaware in that situation, I would fight until one of us cannot fight anymore. I don't think that I would draw at three feet, but I might. If close enough and depending on his size, I may try a redirection and gun grab. Action beats reaction. At that close and wrestling a knife might be the better choice of weapon. No since in introducing another pistol to fight over when that close. Of course I might go for the old draw the wallet and or throw it trick while getting off the X and throwing lead. Should be an easy target to hit. Overall, there is no clear cut answer, except that this punk is threatening to kill me by just having a gun in his hand and that I am not willing chance his mercy.


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Nokia is back! The first new phones from the former number 1 mobile manufacturer now built by HMD, are out. These are not smart phones though, rather new feature phones that gives a glimpse of what HMD can do.
Meet the Nokia 150 and Nokia 150 Dual Sim:
Nokia 150 and 150 Dual SIM specs:
2.4-inch QVGA TFT display (240 x 320 pixels)
Expandable storage via microSD card, up to 32GB
VGA rear camera w/ LED flash
Bluetooth 3.0
FM radio
MP3 player
1020mAh removable Li-Ion battery (BL-5C)
Nokia Series 30+
118.0 x 50.2 x 13.5 mm
81 g
It has an MP3 player, built-in radio, Bluetooth and a VGA camera. The best part, it has a battery life of 22 hours!
Priced at $26 USD or roughly Php1,300 before taxes, the new phones will be available early next year.
Soon though, we'll see the new smart phones from Nokia, I don't know about you but I'm feeling a bit of excitement myself. Check out HMD's website for more info about these new feature phones.


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The 30-minute Perk-Me-Up facial works to refresh skin and diminish signs of aging (a $65 value). A light massage of the face, décolletage, neck, shoulders, and arms also helps relieve stress. The foot-therapy treatment simultaneously works to relieve tension in the lower body with a leg-and-foot exfoliation, massage, and choice of moisturizing or refreshing mask (a $50 value).
Atlantis Beauty Spa
Atlantis Beauty Spa draws inspiration from its namesake city, the fabled underwater paradise. It's chief aesthetician, Magdalena brings more than 40 years of beauty experience and 15 years of teaching aesthetics to the Spa. The spa's staff also bring extensive beautification training that spans from Europe and Canada to the tranquil facility, awash in the glow of flickering candles. Aestheticians employ Scandinavian-style relaxation techniques that include palliative kneads and recordings of lullabies sung by local Vikings. Meanwhile, skin experts incorporate European formulas and touch techniques into facials and body scrubs, designed to combat the effects of aging. The spa also hosts pedicures and massages, as masseuse Adolfo specializes in Chinese, shiatsu, Thai, and deep-tissue modalities.
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The Trail's End Rain Gear Pants by Natural Gear are perfect for every hunt, rain or shine. We all know how early season opening days can be. It may be warm and wet, cold and dry or a combination of many climates. These pants are extremely versatile and packable. Use it as warm weather rain gear, light weight wader pants or an outer shell to go over several layers. Shell is soft, waterproof and breathable. Mesh lining is comfortable, slides on easy and doesn't bind. The pants have an elastic waistband and more. Available in Natural Gear.
Mack's Prairie Wings is not responsible for typographical errors in pricing or product specification inaccuracies in our web site. Prices are subject to change without notice.
Copyright © 2017 - Mack's Sport Shop LLLP - All rights reserved. Site Developed and Maintained by Ability Commerce
Copyright © 2004-2017 - Mack's Prairie Wings, Inc. - All rights reserved. Site Developed and Maintained by Ability Commerce


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Jo Wilfried Tsonga vs Sam Querrey | Monte Carlo 2015 New Jo Wilfried Tsonga vs Sam Querrey Shock?
- published: 12 Apr 2015
- views: 228
Jo Wilfried Tsonga vs Sam Querrey may come to realization during the Monte Carlo 2015 Masters. This is appears to be an interesting match because it would not be the first time that Tsonga and Querrey battled played against each other. Furthermore, the very last encounters between the two players had one of the player winning in series. You may check this video for more info on who of the two won the last Jo Wilfried Tsonga vs Sam Querrey plays. The date of their new match in Monaco is also included in this video. Do you think that Sam Wawrinka may win once more these masters? Please share your opinion on the following website and find an offer for a gift card:http://www.montecarlomasters2015.blogspot.com...


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This subportal is part of the USA Portal.
Texas is the second most populous and the second most extensive of the 50 United States, and the most extensive state of the 48 contiguous United States. The name, based on the Caddo word "Tejas" meaning "friends" or "allies", was applied by the Spanish to the Caddo themselves and to the region of their settlement in East Texas.
Notable peoples from Texas
See: Texas Project Profiles.


text: It is robust plastic outside and aluminum inside Lightweight and aerodynamically shaped for minimal drag For street riders, it can also be a good safety addition to against bumping into car mirrors or other road hazards. This guard protects your front brake from accidentally being engaged if you bump into another rider or other object. Adjustable length and angle for different lever type and adjustment


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Dogs Trust rescue centres receive two puppies for rehoming just days after they were bought as Christmas presents.
The phrase "A dog is for life, not just for Christmas" is
well-known, but sadly this did not apply for puppies Cluedo and Scrabble who were handed in to Dogs Trust rehoming centres on the 28th December 2011.
During the festive period, staff at Dogs Trust's 17 Rehoming Centres across the UK were busy working around the clock caring for Christmas canine casualties.
Dogs Trust Leeds had a 14-week-old Greyhound crossbreed puppy brought into the rehoming centre on 28th December. His owners purchased him on Christmas Eve, but after a few days decided to give him up as he was not yet housetrained and began chewing the children's other Christmas presents.
Dogs Trust Leeds Manager Amanda Sands said: "We have renamed this little pup Scrabble and hope to find him a new home in January with a family that have the time to train a puppy. Scrabble was bought as a Christmas present and his owners did not realise how much time is involved in looking after a puppy – unlike toys they don't come with an
off switch and puppies certainly need lots of training."
Cluedo was also brought in on the 28th December to Dogs Trust Canterbury. The eight-week-old Yorkshire Terrier crossbreed was handed in after an elderly lady walking her dog in the woods was approached by another woman trying to sell the young puppy to passers-by.
Dogs Trust Canterbury Manager Adam Levy said: "We believe little Cluedo was an unwanted Christmas present and his owner was hoping that dog owners walking their pets around the woods would take pity on him and buy him. If no one bought him, the owner said she would dump him in the woods so we are grateful to the elderly lady who brought him into us."
Research* from Dogs Trust revealed that more than a third of children asked for a puppy as a present and 16 per cent of parents would have bought one if their child put it on their Christmas list.
Scrabble and Cluedo are now in safe hands and will be looking for new homes in the New Year. Anyone interested in rehoming the puppies should contact Dogs Trust Canterbury for Cluedo on 01227 792 505 or Dogs Trust Leeds for Scrabble on 0113 281 4920.
For further information on Dogs Trust, visit www.dogstrust.org.uk
*Dogs Trust Survey carried out by One Poll in October 2011. 1,000 respondents of parents with children aged 3 to 16 years old.


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Ryan Johnson's time with the San Jose Earthquakes has come to a close.
The Earthquakes traded Johnson, along with allocation money and an international player spot, to Toronto FC in exchange for Alan Gordan, Nana Attakora and Jacob Peterson in a blockbuster deal on Thursday.
The move provides San Jose with depth at three positions, though it loses an international spot until Jan. 1, 2013.
The trade was Toronto's second of the day, having acquired midfielder Terry Dunfield in a deal with the Vancouver Whitecaps earlier.
What do you think of this trade? Who do you think got the better end of the deal? Surprised Toronto FC made two moves on the same day?
Share your thoughts below.
got to say, you are right on. attakora will be a loss, b/c he has room to grow and i could see him being a very good player in this league. as for JP and AG, typical MLS journey. servicable, you need guys like this to fill out a roster, not be the key components to it.
now TFC adds, johnson and dunfield, potential for a new DP centre half hopefully, and if they ever dump de guzman even more cap space to fill wholes. the team is starting to come together nicely, 2012 will be a big year for them if they can make that next step to perennial play off team
Ossington Mental Youth
I said the samething when he was coming in but he really has played very well (GREAT) this year. i know he was pretty meh in LA and barely played in Chivas but dude was coming into his own here
Ossington Mental Youth
Hes out for the year with an injury.
Unfortunate, curious to see if hes around next year, kinda feel like itll be his swansong
Ossington Mental Youth
4 goals in 8 games, not to mention several canadian cup goals. Once again, effective WHEN hes healthy.
frank from sf
i'm a 'quakes fan and i can tell you, we just traded crap for crap. really no other way around it.
Don't forget Conor Casey – he played two games for Toronto before being shipped off.
Earthquakes Fans Shaken Up
Ryan Johnson works harder than anyone and is a really good player, but he has had a curse hanging over him in terms of finishing for the San Jose Earthquakes. I think more than anything this may be a chance for Ryan to join another club where he can break out, and it could not happen for a better guy.
Not sure why the Quakes are bringing in more depth, a cutting edge would seem to be more what they need.


text:
PENETRON Around the World
Over the past 40 years, the PENETRON system of products has helped our customers undertake and complete ambitious and challenging projects – from hospitals to parking garages, bridges to tunnels, water treatment plants to military installations… and on and on.
To get a closer look at some of our projects, simply click on a project or choose a country or specific category below.
Please contact your PENETRON representative if you need additional project details.
You will learn how PENETRON enables successful projects around the world.
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"W" Residences at Sentosa Cove
The Residences at "W" Singapore is a luxury development that incorporates retail outlets, a world-class…
2014 Winter Olympics
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The 2014 Winter Olympics was the largest single infrastructure project undertaken in Russian history.…
6th of May Park & Dolphinarium
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Located on the shores of the Black Sea, the expansive "6th of May Park" includes a water park (with…
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Fort Lauderdale, FL, USA
The 8th Avenue Residences complex is a cornerstone of the urban mixed-use development in downtown Fort…
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A 33-floor office tower with a 3-floor basement car park, the A&B Tower is located in the heart of Ho…
A-148 Connector Road
Adler (Sochi)-Gornaya Karusel, Krasnodar Krai, Russia
The A-148 mountain highway is a 50 km (32-mile) curvy road that connects Adler, on the Black Sea coast,…
A-370 Amur Bay Bridge
Vladivostok, Russia
The low-lying beam bridge spanning Amur Bay is a four-lane highway that connects the De Friz and Muravyov…
ABB Consortium
Lipetsk, Russia
ABB Consortium opened its first production facility in Lipetsk Free Economic Zone in 2014. The factory…
Adelaide Building
SALVADOR, Brazil
The Adelaide Building was built on the shores ofBaia de Todos os Santos, located in Salvador, which is…
Adler Railway Station
Sochi, Russia
The Adler Railway Station in Sochi – one of the largest railway terminals in Russia – serves the…


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American Made
Make it your lucky day! Whether you're at the track or in the office, this tie will not only guarantee you good fortune, but ensure that you look great while doing it.
Details & Features:
Packaging doubles as a travel case for up to two bow ties
Care Instructions:
If you have any questions, please dont hesitate to call us! 1-804-756-6800, We are here to help.
© Copyright 2017 Collared Greens. All Rights Reserved.


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Staff Pick
We're committed to uncompromising craftsmanship and to your satisfaction. If you are unsatisfied for any reason, return your purchase within 60 days with proof of purchase to receive an exchange, repair, or refund. It's our promise.
Our exclusive blend of Organic cotton, Merino wool and spandex isn't just for long pants anymore. The clean drape of Synergy makes movement easy. Softness, comfort and the moisture management of wool stretch the boundaries of what was possible with previous fabrics. Grab the Synergy short and focus on your own boundary stretching.
Ibex exclusive triple knit jersey. 49% Organic Cotton, 48% New Zealand Merino Wool, 3% Lycra. 280 g/m2. GOTS certified. ZQ Merino Wool.
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text:
Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat
- First Online:
- Received:
- Revised:
- Accepted:
DOI: 10.1186/bcr3192
- Cite this article as:
- Tate, C.R., Rhodes, L.V., Segar, H.C. et al. Breast Cancer Res (2012) 14: R79. doi:10.1186/bcr3192
- Citations
- 46k Downloads
Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo.
TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology.
Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype.
This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.
KeywordsPanobinostat LBH589 triple-negative breast cancer xenograft histone deacetylase inhibitor E-cadherin CDH1 epithelial-to-mesenchymal transition
analysis of variance
- BT-549-tRFP
BT-549 turbo red transfectant
cadherin-1, E-cadherin
Dulbecco's modified Eagle's medium with 10% fetal bovine serum
dimethyl sulfoxide
enzyme-linked immunosorbent assay
epithelial-to-mesenchymal transition
estrogen receptor
histone deacetylase
- HDACi
histone deacetylase inhibitor
hematoxylin and eosin
- Her2/neu
human epidermal growth factor receptor 2
- i.p.
- MDA-MB-231-tRFP
MDA-MB-231 turbo red transfectant
metallothionein 3
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
phosphate-buffered saline
progesterone receptor
- qPCR
quantitative reverse transcription real-time polymerase chain reaction
severe combined immunodeficiency
standard error of the mean
triple-negative breast cancer
- tRFP
turbo red fluorescent protein.
Over 200,000 new cases of invasive breast cancer are diagnosed in the United States each year and approximately 40,000 of the patients diagnosed will die from the disease [1]. Breast cancers are routinely classified by stage, pathology, grade and expression of estrogen receptor (ER), progesterone receptor (PR) or human epidermal growth factor receptor (Her2/neu). Current successful therapies include hormone-based agents that directly target these receptors [2, 3]. Triple-negative breast cancer (TNBC) is a heterogeneous subset of neoplasms that is defined by the absence of these targets [4, 5, 6]. Approximately 15% of globally diagnosed breast cancers are designated as ER-, PR- and Her2/neu-negative [1, 7, 8]. Studies have shown that tumors of this aggressive subtype are of higher histological grade, affect a disproportionate number of young women, and are more likely to recur earlier at distant sites, resulting in poor overall prognoses [4, 5, 9, 10]. To improve outcomes of TNBC, we must unravel its biological pathways and modes of progression and use that knowledge to develop novel targets and therapies.
Histone deacetylase inhibitors (HDACis) have emerged as a promising new class of multifunctional anticancer agents [11, 12]. That promise lies in the ability of HDACis to effect multiple epigenetic changes in aberrant cells. In addition to regulating gene expression and transcription through chromatin remodeling, HDACis can also modulate a variety of cellular functions including growth, differentiation, and survival [13, 14] due, in part, to their ability to enhance acetylation of a wide range of proteins, including transcription factors, molecular chaperones, and structural components [11, 15, 16]. Specifically, HDACis have been linked to several downstream effects in tumor cell lines which include: cell cycle arrest, induction of apoptosis, inhibition of angiogenesis, activation or inactivation of tumor suppressor genes or oncogenes, and decreased invasion and metastases [11, 12, 17].
Panobinostat (LBH589) is a potent pan-deacetylase inhibitor that can block multiple cancer related pathways and reverse epigenetic events implicated in cancer progression [18]. HDACs can be subdivided into two groups: zinc-dependent (Class I, II, and IV) and zinc-independent (Class III) [19]. Panobinostat is a potent inhibitor with activity against Class I, II, and IV HDAC enzymes, suggesting true pan-HDAC activity [18]. In preclinical studies, panobinostat has shown potent inhibitory activity at low nanomolar concentrations across a wide range of hematologic malignancies including lymphoma, multiple myeloma and acute myeloid leukemia [20, 21, 22]. It is also being investigated as a treatment against non-responsive solid tumors as well as tumors of the lung, thyroid, and prostate [23, 24, 25, 26]. It has shown synergy with chemotherapeutics, radiation, demethylators, proteasome inhibitors and other agents [27, 28, 29]. Based on these preclinical findings, panobinostat and other HDACis have undergone a rapid phase of clinical development with many entering clinical trials, both as single agents or in combination with other therapies [12, 23, 30, 31]. To date, panobinostat has demonstrated favorable clinical responses, with limited toxicity [23, 32, 33]. There is a critical need to develop pleiotropic therapies that specifically target the neoplasm as well as the biological pathways and markers of TNBC progression. The purpose of this study was to determine the ability of panobinostat to selectively target the TNBC subtype of breast cancer cells, assessed by its effects on the growth, survival, and tumorigenesis of a representative panel of TNBC cells. We also sought to characterize the effects panobinostat on the regulation of breast cancer genes, related signaling pathways and morphology.
Materials and methods
Cell lines and reagents
Human TNBC (MDA-MB-157, MDA-MB-231, MDA-MB-468, BT-549), MDA-MB-361 and HEK293T (human embryonic kidney)) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MDA-MB-157, MDA-MB-231, and BT-549 cells are characterized as triple-negative/basal-B mammary carcinoma, while the MDA-MB-468 cells are characterized as triple-negative/basal-A mammary carcinoma. MDA-MB-361 cells are characterized as ER-positive/Progesterone receptor (PgR) negative, luminal mammary carcinoma. Liquid nitrogen stocks were made upon receipt and maintained until the start of each study. MCF-7 cells, characterized as ER-positive/PgR-positive luminal mammary carcinoma, were obtained from frozen stocks routinely used in previous experiments [34]. The ER-positive/PgR-positive ZR-75-1 human epithelial mammary ductal carcinoma cells were a generous gift of Dr. Brian Rowan (Tulane University, New Orleans, LA, USA). Cells were used for no more than six months after being thawed with periodic recording of morphology and doubling times to ensure maintenance of phenotype. Cells were maintained at 37°, 5% CO2 in 10% (D)MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, Salt Lake City, UT, USA) and 1% penicillin/streptomycin (Invitrogen). Panobinostat was generously provided by Novartis Pharmaceutical Inc. (East Hanover, NJ, USA). Panobinostat was dissolved in dimethyl sulfoxide (DMSO) (Invitrogen) as a 1 mM stock solution and kept at -20°C. The drug was diluted in culture media and used at various concentrations as indicated.
Histone acetylation
TNBC cells were plated at 70% confluency in 10% (D)MEM and allowed to attach overnight. Cells were treated with panobinostat (100 nM, 200 nM) or vehicle for 18 hours, then fixed, permeabilized and stained with acetyl-histone H3 (Lys9) antibody/Alexa Fluor® 488 Conjugate or acetyl-histone H4 (Lys8) antibody/Alexa Fluor® 488 Conjugate (Cell Signaling Technology, Danvers, MA, USA), followed by rhodamine phalloidin and DAPI (4',6-diamidino-2-phenylindole) counterstain (Molecular Probes, Carlsbad, CA, USA) according to the manufacturer's instructions. Cells were dually analyzed by BD LSR II flow cytometer and BD Pathway 855 bioimaging confocal system and images merged using BD Attovision™ Software (BD Biosciences, San Jose, CA, USA). Data are represented as mean fluorescence intensity (mean ± SEM) of two independent experiments with internal triplicates.
MTT cell proliferation assay
Proliferation was measured by MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Cell Proliferation Assay, according to the manufacturer's protocol (ATCC). Briefly, cells were plated in 96-well flat bottom plates at a density of 5 × 103 per 100 μl in 10% (D)MEM, allowed to attach overnight, and then treated with panobinostat (50, 100, 200 nM) or vehicle for 24 hours. MTT reagent (10 μl) was added to each well (final concentration 0.5 mg/ml) and the plate incubated at 37°C. After four hours, 100 μl of solubilization solution (10% SDS) was added to each well and the plate incubated for two hours. A matched control cell standard curve using sequentially increased cell numbers was included on the plate for each corresponding cell line to determine growth inhibition. The absorbance was read at 570 nm on a Synergy™ 4 Multi-Mode Microplate Reader and analyzed with Gen5™ Data Analysis Software (BioTek, Winooski, VT, USA). Data are represented as mean percent vehicle treated cell proliferation ± SEM of triplicate experiments with internal triplicates.
Trypan blue viability assay
Cells were plated in 96-well plates at a density of 5 × 103 per 100 μl in 10% (D)MEM and allowed to adhere overnight. Cells were treated with vehicle or panobinostat (50, 100, 200 nM) for 24 hours and harvested by trypsinization. Cells were then stained with a trypan blue solution (0.04% w/v, Invitrogen), and counted on a Cellometer Vision automated cell counter (Nexcelom Bioscience, Lawrence, MA, USA) according to the manufacturer's protocol. Cell viabilities are represented as mean percent relative to matched, vehicle-treated cells ± SEM of triplicate experiments with internal triplicates.
Analysis of apoptosis was carried out using the Cell Death Detection ELISAPLUS according to the manufacture's protocol (Roche Applied Science, Mannheim, Germany). This quantitative DNA fragmentation immunoassay uses monoclonal antibodies directed against histone-complexed DNA. Briefly, cells (104 cells/well) were plated in 96 well plates overnight and treated for 24 hours with panobinostat (100, 200 nM) or vehicle control. After cell lysis and centrifugation, the cell lysates were tested for histone-complexed DNA fragments. The absorbance was read at 405 nm on a Synergy™ HT Multi-Mode Microplate Reader and analyzed with Gen5™ Data Analysis Software (Bio-Tek). Apoptosis of the treated cells was expressed as mean enrichment factor (treated cells over vehicle controls) ± SEM of duplicate experiments with internal triplicates.
Cell cycle analysis
For cell cycle analysis, TNBC cells were plated overnight in 10% (D)MEM and treated with 100 nM panobinostat for 24 or 72 hours. Both floating cells and trypsinized adherent cells were collected and combined for analysis. Cells were fixed by dropwise addition into ice cold ethanol and stored at -20° overnight. Cells were then pelleted, washed, and stained for one hour with 50 μg/ml propidium iodide in PBS containing 0.1 mg/ml ribonuclease A and 0.05% Triton X-100 (Sigma, St. Louis, MO, USA). After gating to exclude debris, the DNA content was measured using a LSR-II flow cytometer (BD Biosciences). Data were analyzed with ModFit LT software (Verity Software House, Topsham, ME, USA). Data are represented as percent live cells of two independent experiments.
Plasmid packaging and stable cell line generation
HEK293T cells were plated at 5.5 × 106 in a 10 cm dish in 10 ml of 10% (D)MEM and allowed to adhere overnight. The following day, the HEK293t cells were co-transfected with the pLEmiR non-silencing turbo red fluorescent protein (tRFP) vector construct (9 μg) and the trans-lentiviral packaging mix and pLEX™ transfer vector using the TransLenti Viral pLEX packaging system, following the manufacturer's instructions (Thermo Scientific, Waltham, MA, USA). Virus was harvested 48 hours post-transfection and stored at -80°C. TNBC cell lines were plated at 70% confluence in 10 cm dishes with 10 ml of 10% (D)MEM and allowed to adhere overnight. The following day, cells were transduced with virus containing the pLEmiR tRFP vector (1:10 dilution) in serum-free media following the manufacturer's protocol. After four hours, the transduction media was removed and replaced with 10% (D)MEM. After 24 hours, cells were treated with puromycin (Invitrogen) to select for vector expression. The resultant stable transfectants were designated as MDA-MB-231-tRFP and BT-549-tRFP.
Animal xenograft studies
Xenograft tumor studies were conducted as previously described [34]. In short, CB-17/SCID female mice (four to six weeks old) were obtained from Charles River Laboratories (Wilmington, MA, USA). The animals were allowed a period of adaptation in a sterile and pathogen-free environment with food and water ad libitum. MDA-MB-231-tRFP and BT-549-tRFP cells were harvested in the exponential growth phase using a PBS/ethylenediaminetetraacetic acid (EDTA) solution and washed. Viable cells (5 × 106) in 50 μl of sterile PBS suspension were mixed with 100 μl reduced growth factor Matrigel (BD Biosciences) and injected bilaterally into the inguinal mammary fat pad. On day three post cell injection, mice were randomized into treatment groups of five mice each: (vehicle control or 10 mg/kg panobinostat). Beginning on day 14 post cell injection, animals received intraperitoneal (i.p.) injections of the corresponding drug treatment on a five-day on and two-day off schedule for 28 days [18]. Tumor size was measured with a digital caliper and calculated using the formula 4/3πLS2 (L = larger radius, S = smaller radius). At necropsy, animals were euthanized by cervical dislocation following CO2 exposure. Tumors, livers, lungs, and brains were removed and snap frozen or fixed in 10% formalin for future analysis. All procedures involving animals were conducted in compliance with State and Federal laws, the U.S. Department of Health and Human Services, and guidelines established by Tulane University Animal Care and Use Committee. The facilities and laboratory animals programs of Tulane University are accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care.
Human breast cancer quantitative reverse transcription real-time PCR array
Human Breast Cancer and Estrogen Receptor Signaling RT2 Profiler™ PCR Arrays (PAHS-005) were obtained from SABiosciences (Frederick, MD, USA). MDA-MB-231, MCF-7, and MDA-MB-468 cells were plated in 10% (D)MEM at 70% confluency and treated with 100 nM panobinostat or vehicle for 24 hours. Cells were harvested by trypsinization and total RNA was isolated using the RNeasy kit, according to the manufacturer's instructions (Qiagen, Valencia, CA, USA). The quantity and quality of the RNA were determined by absorbance at 260 and 280 nm using the NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). Total RNA (1.5 μg) was reverse-transcribed using the RT2 First Strand cDNA synthesis kit, following the manufacturer's protocol (SABiosciences) and then assayed via an optimized, quantitative RT real-time PCR (qPCR) array to assess panobinostat-associated changes in the expression of 84 genes related to breast cancer regulation and estrogen receptor-dependent signal transduction, according to the manufacturer's protocol. Biological triplicates were run for each sample.
CDH1 flow cytometry and immunofluorescent imaging
MDA-MB-231 cells were plated at 70% confluency in 10% (D)MEM and allowed to attach overnight. Cells were then treated with panobinostat (100 nM) or vehicle for 24 hours. Cells were harvested by gentle pipetting (PBS with 5% fetal bovine serum), fixed, and stained with Alexa Fluor® 488-conjugated CDH1 (E-cadherin) antibody (BD Biosciences). The expression of CDH1 protein was determined by flow cytometry on a BD LSRII instrument. Data are represented as mean percent E-cadherin positive cells ± SEM of duplicate experiments with internal triplicates. Paired cells were seeded on BD Falcon 96-well black imaging plates. Staining is represented by the following colors: Green = CDH1, Red = phaloidin, Blue = DAPI nuclear stain. Confocal immunofluorescent images were captured on the BD Pathway 855 Bioimaging system and merged using BD Attovision™ software (BD Biosciences).
ELISA for CDH1
MDA-MB-231 cells (105 cells/well) were plated overnight in six-well plates and then treated for 24 hours with panobinostat (100 nM) or vehicle control. Five volumes of ice-cold lysis buffer (20 mM Tris-HCl, pH 7.5/150 mM NaCl/1 mM EDTA/1 mM ethylene glycol-bis[β-aminoethyl ether]-N, N, N', N'-tetraacetic acid (EGTA)/1% Tween 20) supplemented with protease inhibitor tablets (Roche Diagnostics, Indianapolis, IN, USA) were added to each well. Cell lysates were mechanically dissociated and centrifuged (10,000 × g for 15 minutes at 4°C), and then diluted 1:1 with calibrator diluent. CDH1 levels were then determined by human CDH1 ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). The absorbance was read at 450 nm on a Synergy™ HT Multi-Mode Microplate Reader (Bio-Tek). Data are represented as mean pg/ml of CDH1 ± SEM of duplicate experiments with internal triplicates.
Immunohistochemical staining
Representative sections of tumor with adjacent tissues were fixed in 10% neutral buffered formalin for 24 to 36 hours. Paraffin-embedded sections were prepared at 4 μm thickness followed by standard H & E staining. Additional sections were manually deparaffinized in xylene, rehydrated in a series of graded ethanol solutions, boiled in10 mM sodium citrate buffer (pH 6.0) for ten minutes, then cooled for 20 minutes for antigen retrieval. Sections were blocked for 30 minutes with 10% normal goat serum (Invitrogen), incubated overnight in a 4° humidified chamber with rabbit anti-E-cadherin (Abcam, Cambridge, UK) at 1:30 dilution, followed by one hour incubation with Alexa Fluor® 488 goat anti-rabbit secondary (Invitrogen). Fluorescent images were captured on a Nikon TE2000 inverted microscope with IPLab software (BD Biosciences, Rockville, MD, USA).
Statistical analyses
Statistical analyses were carried out with GraphPad Prism software (Graph-Pad Software, Inc., San Diego, CA, USA). Studies involving more than two groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc multiple comparison tests. All others were subjected to unpaired Student's t-test, with P < 0.05 considered statistically significant.
Panobinostat induces histone acetylation
Panobinostat cytotoxicity in TNBC cell lines
Effect of 100 nM panobinostat on cell cycle percentage of TNBC cells.
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Panobinostat targets tumor growth in vivo
Panobinostat regulates breast cancer genes and estrogen signaling pathways
To reveal possible molecular mechanisms and signaling pathways involved in TNBC cell response to panobinostat, MDA-MB-231 cells were treated for 24 hours and analyzed with the Human Breast Cancer and Estrogen Receptor Signaling RT2 Profiler™ PCR Array (SABiosciences). As shown in Additional file 1, thirty-five of the eighty-four representative genes were significantly altered at least two-fold (P < 0.05). Specifically, expression of twenty-four genes was up-regulated while expression of eleven genes was suppressed. Of particular interest was the 31-fold increase in the documented epithelial cell marker/tumor suppressor, CDH1 [35]. Also noted were decreases in the proliferation marker MKI67 and upregulation of the tight-junction protein, claudin-7.
Panobinostat induces changes in morphology and CDH-1 expression of MDA-MB-231 cells consistent with reversal of EMT
In recent years, an increasing number of HDACis have been identified, developed and advanced to clinical trials [39, 40]. Panobinostat has shown potent activity at low nanomolar concentrations across a wide range of hematologic malignancies and solid tumors in preclinical studies [20, 21, 22, 41]. Others have demonstrated that HDACi treatment can suppress oncogenes and induce re-expression of previously silenced tumor suppressors and receptors such as the ER [24, 42, 43, 44]. In addition to its single agent effects, recent studies have demonstrated a role for panobinostat in resensitizing cancer cells to other agents including chemotherapy [45], radiation [46], autophagy inhibitors [47] and endocrine therapies including tamoxifen [48] and letrozole [49]. In consideration of the promising results reported by others, we endeavored to determine whether panobinostat would be effective against a panel of breast cancer cell lines that display common characteristics of the triple-negative subtype.
In this study, we utilized MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT549 cell lines as models of TNBC growth and progression. In confirmation of other preclinical research [20, 24, 42, 44, 50, 51], we found that panobinostat induced hyperacetylation of histones H3 and H4, decreased proliferation and survival, and induced apoptosis and G2/M cell cycle arrest. The MDA-MB-231 and BT549 lines were chosen as models for our in vivo xenograft studies using CB-17/SCID mice. Treatment with panobinostat decreased MDA-MB-231 and BT549 tumor significantly with minimal animal toxicity, providing preclinical data on the effectiveness of panobinostat on TNBC tumorigenesis at a low and well tolerated dose.
The panobinostat-induced effects on cell proliferation and survival appear to be TNBC cell specific as the ER-positive cell lines tested were unaffected at all doses tested (up to 200 nM), contrary to previously published work which reported panobinostat significantly inhibited cell survival and induced cell death in ER-positive and ER-negative breast cancer cell lines though at a different time point [44, 47]. We propose that the more aggressive, highly proliferative nature and invasive phenotype of TNBC cells render them particularly susceptible to the effects of panobinostat. Of the four TNBC cell lines tested, the MDA-MB-468 cells were the most resistant to hyper-acetylation and DNA degradation by the drug. This is interesting as this cell line is the most phenotypically different (spherical morphology) and least invasive of the four tested cell lines. The MDA-MB-157, MDA-MB-231, and BT549 lines have been classified as basal-B [52], with the MDA-MB-231 and BT-549 cell lines specifically classified as mesenchymal (stellate), claudin-low, and highly invasive [53, 54, 55, 56]. The MDA-MB-157 cells are classified as mesenchymal, claudin-low, and moderately invasive [52]. Clinically, the majority of claudin-low tumors are of the triple-negative subtype and are associated with poor overall prognoses [53]. However, MDA-MB-468 cells have been characterized under the basal-A subtype, as they possess both basal (triple-negative) and luminal (spherical morphology) characteristics and are only minimally invasive [52]. Additionally, super array data comparing panobinostat-induced gene expression changes between panobinostat-sensitive (MDA-MB-231, basal-B) and panobinostat-insensitive (MDA-MB-468, basal-A; MCF-7, luminal) cells revealed several changes specific to the basal-B subtype [See bolded genes in Additional file 1]. These ten genes include known regulators of cell proliferation (FOSL1, STC2, TGFA, THBS2) and apoptosis (FAS, FASLG), as well as angiogenesis (TNFAIP2). Additionally, many of the genes altered by panobinostat specifically in MDA-MB-231 cells have documented roles in cell invasion and metastasis including CDH1, CLDN7, FOSL1, PLAU, STC2, and TGFA. These data support the role of the selective effects of panobinostat observed on the basal-B cell lines compared to the other subtypes tested.
Interestingly, superarray data identified CDH1 as being the most induced gene by panobinostat treatment specifically in MDA-MB-231 cells, as these cells are characterized as mesenchymal, thus lacking significant CDH1 expression. The TNBC subtype is exemplified by its highly aggressive and metastatic nature. A known key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). This oncogenic EMT is typified by increased invasion and metastatic dissemination, therapeutic resistance and loss of expression of tumor suppressors such as CDH1 [57, 58]. Studies have demonstrated that EMT and the resultant loss of CDH1 expression are crucial steps in tumor progression and correlate with poor clinical outcomes [59, 60, 61]. In confirmation of our in vitro data on CDH1 up-regulation, we also noted an increase in CDH1 on the periphery of the primary tumor from our MDA-MB-231 xenograft model. Decreased CDH1 expression at the tumor periphery has been linked to increased metastasis-risk and decreased overall patient survival [62]. Induction of CDH1 expression by LHB589 at the invasive edge may therefore be indicative of decreased metastatic potential. Panobinostat-induced re-expression of CDH1, along with other morphological features, indicates the partial reversal of EMT, a target of enormous potential, particularly in the TNBC subtype. This suggests panobinostat as a promising therapeutic option for the more aggressive, TNBC/basal-like breast cancer subtypes.
Our results illustrate the ability of panobinostat to hyperacetylate histones, inhibit proliferation and survival, and decrease in vivo tumorigenesis of TNBC cells. Our in vitro data suggest that this cytotoxicity is partially due to cell cycle arrest and apoptosis. Also noted in treated cultures was an apparent partial reversal of the mesenchymal phenotype evidenced by increased CDH1 protein expression and morphology changes in MDA-MB-231 cells. This increased CDH1 was confirmed with measured upregulation of the CDH1 staining at the primary tumor periphery in our xenograft model. Overall, our results affirm the efficacy and demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.
The authors would like to acknowledge Dr. Kenneth Nephew and Dr. Brian Rowan for critical review of this manuscript. We also acknowledge the expert skills and advice of Mary Price and the Louisiana Cancer Research Consortium FACS Core Facility. This research was supported by: Susan G. Komen Breast Cancer Foundation BCTR0601198 (ME Burow); The Department of Defense Breast Cancer Research Program BC085426 (BM Collins-Burow); The National Institutes of Health/National Center for Research Resources P20RR020152 (BM Collins-Burow) and CA125806 (ME Burow). The funders did not have any involvement in study design; the collection, analysis, or interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication.
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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