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In Stitches!
Latest Posts
6 Wet Felting Projects I'd Like to Try
Making felt from wool fibre is a magical thing. You start with a masses of soft fluffy roving that resemble fairy floss, and through the wonderful process of felting, the fibres that once barely touched each other are bonded for life in a strong compact fabric. It's quite the transformation. I've enjoyed the craft of needle felting for many years…Read More
Upcycle Style: Crochet T-shirt Rag Rug
After making a handy crochet basket from t-shirt yarn last week I was itching to experiment with some homemade t-shirt yarn and made a Crochet Rag Rug from some old clothes that I had laying around. I took the opportunity to make a video of whole the process and included some instructions for changing yarn colours and how to do…Read More
Crochet Pattern: Rainbow Storage Basket
I love crocheting with t-shirt yarn, it works up so quickly and creates a very robust fabric which is perfect for decor items that need to stand up to wear, like storage baskets and rugs. I have accumulated quite a stash of t-shirt yarn, and because it's bulky storage is always tricky. The perfect solution to my yarn storage dilemma…Read More
How To Sew an Easy Zippered Pillow Cover
I've been going through my blog archives, tidying up images, old links and broken code. It occurred to me that I've got a lot of great tutorials buried deep in those old archives which you've probably never seen, so I've decided to occasionally republish the best of them. This week I'm re-sharing the instructions for making a simple zippered cushion…Read More
DIY Princess Bubblegum Crown
We are heading to Oz Comic-Con tomorrow. We had a fun time last year when Emma dressed as Raven from Teen Titans, and after many, many weeks of deliberation, she finally settled on Princess Bubblegum from Adventure Time. Princess Bubblegum (PB for short) is made of bubblegum (hence the pink), and is the ruler of the candy kingdom. Not just…Read More
Beaded Fairy Bread Sew-on Patches
Sew-on patches are so hot right now. Forget the boring old scout patches from back in the day (although I must say, I still think they are pretty cool), patches are getting whimsical, colourful and fun. I made some fairy bread socks for a project in Frankie Magazine, and thought you may like to make your own fairy bread patches…Read More
Put your best foot forward – Frankie Magazine Sock Auction
If you've had a chance to spy the latest issue of Frankie magazine, you would have seen their latest collaboration with 10 talented ladies to transform an ordinary pair of socks into something worth kicking up your heels for. The Sock Project aims to raise funds for the Australian Literacy and Numeracy Foundation by auctioning off the wearable works of…Read More
3D Printing & Resin Decorating Workshop with Mimaw + Etsy
Last week I had the pleasure of joining the Melbourne Etsy team in the Footscray studios of MIMAW, a 3D printing and design practice. It was a fascinating insight into not only the physical process of making a 3D printed object, but also all the design and work that is required behind the scenes before you even turn the printing…Read More
How to Make: Melt-and-Pour Soap
Have you ever tried making soap? It looks really fun and they make wonderful gifts. This soap making project is by Michelle Mackintosh from her new book Care Packages. Making soap the traditional way is a bit of a tricky business, and not a suitable activity for children due to the use of caustic soda. However, making soap from a…Read More
Let's Talk about Mending
In this day and age where it's seems easier to just go out and buy new things rather than repairing what we already have, mending is becoming a lost art. With the rise of fast fashion and built in obsolescence, fixing what's broken can seem unnecessary and in some cases, due to intentional design, physically impossible. But for many people,…Read More
Book Preview {Care Packages by Michelle Mackintosh}
When you are far from home or feeling a bit under the weather, there's nothing quite like receiving a Care Package to make the day a little brighter. In her new book, Care Packages, Michelle Mackintosh is bringing back the art of crafting packages with love and care. Care packages get their name from the humanitarian organisation Cooperative for Assistance…Read More
80s Vintage Jacket Refashion
I'm pretty excited to finally start tackling my clothing refashion pile and sharing some of my projects with you. I actually do a fair bit of refashioning but because taking photos of myself wearing the items can be a bit tricky, I rarely blog them. Well I intend on changing that, so this week I dragged out the tripod and…Read More
Upcycle Style: Recycled Fabric Twine Planter
I'm always looking for nifty ways to use my recycled handmade scrap fabric twine and today I have a project that not only uses recycled fabric scraps, but an old yoghurt rub as well. I've been holding onto the empty tubs that my favourite yoghurt comes in, because they are the perfect size to store pencils and other random craft…Read More
8 Gorgeous Floral Gifts to Make this Mother's Day
Flowers make the perfect gift for Mother's Day. I mean breakfast in bed is pretty nice, and I do love a good macaroni necklace, but I've yet to meet another mother that doesn't enjoy a nice bunch of flowers. There's just one hitch, florists are run off their feet at this time of year, so why not get creative and…Read More
12 Unique Gifts that Crafty Mum's will Love – Mother's Day Gift Guide
If you are looking for a special gift for your craft loving Mum, Nanna, (or even yourself) this Mother's Day, look no further. I have scoured Etsy for colourful, practical and very unique crafty handmade and vintage finds. Trust Etsy Makers to make with Makers in mind, from wearable tokens of affection for yarn, to gorgeous one of a kind…Read More
Colour a Cake – Free Printable Colouring Page
It's Emma's birthday week, and just like every other year for several weeks before her birthday she has been poring over the pages of everyone's favourite cake book, The Australian Women's Weekly Children's Birthday Cake Book. Hands up if you had a swimming pool cake or a dolly varden. If you were a kid in Australia in the 80's you'll…Read More
Fruity Paper Plate Envelopes
It's been a while since I've made something exciting for my pen pals, so this week I got creative with some paper plates and transformed them into colourful Fruity Envelopes. I always try and make their mail deliveries a little bit more fun, like the times I sent a giant hamburger postcard, an ice-cream cone and a Christmas stocking. Fun…Read More
Easy Scandi Inspired Plant Pot Makeover
Until recently I wasn't a fan of indoor plants, I think it all stemmed from my father once telling me you shouldn't have plants in your bedroom because at night they will use up all the oxygen as they respire. Well it's sort of true, but the oxygen they consume is minimal so there is no need for alarm. Phew!…Read More


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—To determine the effectiveness of follow-up tests for signaling recurrences in patients with intermediate- and high-risk malignant melanomas treated with curative intention.
—Retrospective analysis of prospectively collected data.
—North Central Cancer Treatment Group.
—A total of 261 patients with resected local (≥1.69 mm) and regional nodal malignant melanomas who were enrolled in a single prospective adjuvant trial were studied. All patients were scheduled to be followed up monthly for 2 months, then every 2 months for the first year, every 4 months the second year, every 6 months the next 3 years, and annually thereafter, with each visit consisting of a history, physical examination, complete blood cell count, blood chemistry panel, and a chest x-ray.
—Of the 145 evaluable patients who developed recurrent melanomas, 99 patients (68%) developed symptoms that signaled the diagnosis of recurrent disease. Physical examination of asymptomatic patients led to the diagnosis of recurrent disease in 37 patients (26%). The other nine patients (6%) with recurrent disease had abnormal chest x-rays. Laboratory results were never a sole indicator of recurrent disease.
—The majority of recurrences following resection of primary melanomas are discovered by history and/or physical examination despite the frequent use of other follow-up tests. The present data indicate that routine blood analyses and chest x-rays have limited value in the postoperative follow-up of patients with resected intermediate- and high-risk melanomas.(JAMA. 1995;274:1703-1705)
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...couldn't put Little Georgie back together again (forget George III---I meant "George the Sot" and his splendid little war!!!) There are approximately 144K 'boots on the ground' in Iraq (figure another 10x's that in RAMFs (Rear Area MotherFuckers)sustaining the force. If you count the 20K surge (minus Iraqi government forces and allied militia) and don't count the surge in the current 100K Private Contractors in Baghdads---them's a shitload of people for such a small little village (44mile by 44mile). And yet the carnage continues?!!! I wanted to write SEC of State DR. Connie Rice "Why is America so adament about preventing civil war in Iraq? Civil Wars aren't necessarily a bad thing, after all---if it wasn't for the AMERICAN CIVIL WAR, you wouldn't be Secretary of State, now would you, DR RICE!!! Then I'd have to call for the Secret Service to get her off of me!!! They don't call her the 'Hitlady in Heels' for nothing!!!
Last time I was in ATL, I was almost arrested which meant I could have been reading about...me!!! OFFICERS approached a 39 year old male seated in a rental car near Midtown ATL. OFFICERS observed the man playing music and bobbing his head up and down. When approached, suspect was alone in his car and his pants were up. Suspect said he had just purchased several used CDs and was enjoying the music. When informed that he was parked in a location known for Prostitution, suspect replied: "They're right about Southern Hospitality---I just arrived in town 15 minutes ago and already you guys (Cops) are showing me a good time. I swear to God; the cops here are like the Welcome Wagon or something!". Police told suspect "Ya'll come back, now...BUT NOT HERE!!!"
Hey Lauren... How Ya'll all doin' in the Southern Fried State?!!! I could see you as a guest at a 3D Grade Show and Tell. "Hi, my name is Lauren and I am a writer. My job is to get up every morning and write about the good folks of AT-Lanta. The pay is good but my social life---SUCKS!!! Hey, my job is to write about Atlanta---I don't want to run into the people I write about... And REMEMBER, KIDS---double lock your door at home!
I didn't like the tone of the Blotter this time. What makes the blotter interesting is it's stilted, police-like commentary. Please despence with the scarcasm and witty set-asides; the daily 'ATL Freakshow' is funny/scarey enough without it. FIY, the blotter is "my FIRST READ" of Creative Loafing.
I am courious what gives you the strenght to go to work each morning? Think about it. You get up, sip your morning coffee and grab your car keys...for what? So you can write about the sick-o's, druggies, crazies and the rest of the 'human freak show' living outside your locked door? HELLO? Has is occured to you that you live amongst these people? Do you, like, carry a gun and/or extra life insurance? Isn't your life 'an accident or crime statistic about to happen? If it matters, I love reading your colume---and to think I thought the people of San Francisco were 'weird'!!!
I miss your colume!!! The Blotter and News of the Weird were my favorite items in Creative Loafing. FYI, I 'grade' your articles like a 4th Grade School teacher---with a red Le Pen and a self serving smirk. Here are my codes: "FS" (Fuckin' Stupid), "FN" (Fuckin' Nutz), "ATL" (Only in Atlanta or Georgia---like your prohibition against selling Dildos on Sunday), "STAR" (Fuckin' Badge Heavy COP-Suckers), "SH" (Shit Happends) "OG" (Original Gangster=Criminal activity) "NS" (No SHIT!!!) Example, the dipshit cop who left his car keys in his crusier and didn't set the park break (it rolled and hit a pole) rated a STAR, FS and a NS rating. I was tempted to also code it ATL but our cops in SF are just a bad(San Francisco). They will beat up and rob tourists of their Taco Bell Chalupas if they are 'really in the mood for Mexican' it is happends to be the last one sold that night!!! I would love to see your article in the SF Weekly or Bay Area Guardian!!!
Hey, Atlanta... I am Writing Mayor Newsome of San Francisco to get a restraining order against your city for stealing our TVs away from us---if you take all of our TVs away from us, the Tourists will have nothing to gawk at on Polk Street!!! So he-she stole a tiger striped purse worth $4k? Tiger striped? P-L-E-A-S-E!!! Tiger strips are so last year, Honey!!! Besides, how will he-she coordinate it with her shoes---they stopped making Tiger Striped pumps and slingbacks in the 80's!!! Finally, I liked the store's response: "Help, Help, POLICE! We have been robbed---we'll get back to you in four days to file a report!!!


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Founded in 1996 as a 501(c)(3) non-profit organization, we help entrepreneurs launch new businesses, grow them across national borders, and create wealth for their shareholders. We put "global" in global entrepreneurship.
Our Mission is to spread wealth creation around the world by encouraging entrepreneurship and sharing knowledge to those who educate and support entrepreneurs.
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Through our Global Entrepreneurship Program we facilitate introductions to investors, professional service providers, and other entrepreneurs. Using our exclusive "Roadmap to Entrepreneurial Success" we have helped entrepreneurs raise over $100 million.
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Top Definition
The act of partaking in alcoholic beverages on Wednesdays, with or without company. This is proved to make you feel better and help you to make it through the rest of the week. So cheers and happy hump day (wellness Wednesday)!
Me: Hey Tic! Want to come over for wellness Wednesday?
Tic: Sure, I'll bring the Stones!
Tic: Sure, I'll bring the Stones!
by cmb009 October 12, 2010
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Today's Featured Artist: Michael Godard
Pixels Exclusive: Introducing the hottest summer accessory of 2016... round beach towels! FREE WORLDWIDE SHIPPING.
Fine Art America has expanded to 14 fulfillment centers in 5 countries!
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3 Years Ago
I love looking at and photographing gargoyles. They fascinate me. I was looking around FAA the other day and realized there isn't a group devoted solely to gargoyles. So I started one. If you're a gargoyle fan like me, please consider joining.
NB: I've never administered a group on FAA before. For those of you more experienced at that, if I'm doing it wrong, please feel free to send me a private message and let me know about it. Thanks!
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Posted by: Isabella Abbie Shores on 3 Years Ago
3 Years Ago
There is only one way to run a group and that is your way
http://fineartamerica.com/groups/group-and-contest-administrators.html?tab=overview Please join this group
Groups are not just places to store work but a social area for members to meet like minded people and to hold events and discussions among themselves.
A LOT of groups have just been closed due to administrator inactivity. We would like to make sure that all group admins understand what they are taking on when they look after a group. It is a responsibility some people are not prepared for.
* People should not open groups for just ANY type of art but, instead, all groups should have a theme. We already have an area for all art to be uploaded to, it is called FAA.
* Groups are not meant for personal galleries. You have personal galleries and the Groups are for open membership by other members.
* Make sure your requirements are clearly stated on the front of the group. If all members are submitting unsuitable work then look again at how you have worded things in your group rules. Don't chat on your homepage or people will leave not reading the rules. Keep them numbered and clear and concise
We have main forum rules http://fineartamerica.com/showmessages.php?messageid=260080 which your members must abide by also
* Make sure you have time to administer new images at least once a fortnight.
* Do not be afraid to REJECT work. Members will not mind, if you have clearly stated your criteria on the front page of your group.
* Feature artwork by your members on the front page of the group. Members like to feel you care about them and, one way of doing this, is to make sure you feature a certain number of works weekly, fortnightly or even monthly. Keep the number of features small. Under 30 or people will have a problem opening your home page and will just leave
* Run contests from the group. Allow your members to have contests between themselves. Suggestions for prizes can be anything from a feature on the front page of the group, to a feature on your blog here at FAA.
* FAA does not approve of monetary requirements to enter a group or a contest. Groups and Contests that do this, or who demand registration on another site, may be closed.
* As Group admins you have the right to remove a member from your group if they break your rules. You also have the right to remove artwork. However it is your responsibility to make sure members of your groups understand your rules so you must have these clearly marked on your front page.
* Do not send emails to your members more than once a week and ALWAYS link to the group in the email.
These are the Guidelines we have at present for running groups. They may be added to as we get suggestions and I will update here and make sure the thread is bumped if I add to it.
Thank you for being a responsible group administrator
Posted by: Antonin Gauthier on 3 Years Ago
3 Years Ago
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Facebook Mobile 3.0.1
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New Delhi: Accepting one's follies is often the key to achieving great things.
And Delhi opener Shikhar Dhawan doesn't shy away from confessing that a lack of maturity and inconsistency is the prime reason that he hasn't yet taken the leap from the domestic to the international stage successfully.
Speaking to MAIL TODAY, the newly-married Dhawan said that over the years, he has been guilty of not only throwing his wicket away, but also of not scoring big runs on a consistent basis.
"Over the years, I have always ended up throwing my wicket away. I shall say it was my immaturity that I never valued the importance of staying at the crease and making hay while the sun shines," he said.
"I have had opportunities here and there, but I haven't been consistent enough to stake my claim for a permanent spot in the Indian team. You have to score 1,000-1,500 runs in a domestic season on a regular basis."
Dhawan says he realised the importance of being consistent during his tour of West Indies with the national team last year.
"That tour was an eye-opener for me. I scored a fifty in the opening match of the series, but failed to convert it into a three-figure score. I should have scored a couple more fifties and maybe I would have been a part of the ODI team that toured England. But I failed to grasp the opportunity and I haven't received another one since," he said.
But now, interactions with Virender Sehwag have really helped Dhawan cleared his mind.
"I interacted quite a lot with Viru bhai recently and he made one thing very clear to me. He said that every time a batsman goes out to bat, he should think of it as the only opportunity to show the world how good a batsman he is," the 26-year-old left-handed batsman said.
"I feel that has really made me value my wicket and now whenever I go out to the middle, I try and put a heavy price-tag on my wicket."
Being a nationally contracted player, he can train at the National Cricket Academy (NCA) in Bangalore whenever he wants, and he feels training with Yuvraj Singh the last time he was there has also made him realise the importance of self-introspection.
"Yuvi paji was at the NCA when I went there last time and the experience I had was amazing. Having battled cancer himself, he does know what fighting adversity means and all he told me was that we should always sit down and think about the mistakes we are committing," he said.
Dhawan feels need to be more consistent
Dhawan says he realised the importance of being consistent during his tour of West Indies with the national team last year.By Baidurjo Bhose | Mail Today – Fri 9 Nov, 2012 7:32 AM IST
New Delhi: Accepting one's follies is often the key to achieving great things.
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Sasha reports on Llewellyn King's campaign to find a lone Congress member to do the right thing ...
Discuss the article on the Forums.
Anyone else getting brainfog from every antibiotic?
Discussion in 'Antivirals, Antibiotics and Immune Modulators' started by bedman, Jun 18, 2011.
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YouTube clone
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ATV Offroad Fury: Blazin' Trails PSP
- Publisher: SCEA
- Release Date: Apr 19, 2005
- Critic score
- Publication
- By date
Despite unresponsive controls and average graphics riddled with a jerky frame-rate (elements that would normally kill any racing game's enjoyment) Blazin' Trails delivers a surmountable and fun but challenging struggle for first place.
Unfortunately, the control isn't anywhere near at tight as the previous PS2 games, and you'll often find yourself overcompensating and weaving around the track. It doesn't ruin the game, but it makes it harder than it was designed to be.
If you're in love with the grit and grime of dirt racing, and don't mind a challenge, then Blazin' Trails should be right up your alley.
If you're prepared for the steep learning curve and the insane load times, Trails is yet another extremely competent PSP title that's certainly worth checking out.
Jumps are a bit hard to do and some of the more advanced stunts are really a challenge.
Given the much too difficult AI, the pre-load that leaves you anywhere but on track and the ATV being placed in the most annoying locations, gamers that are easily frustrated will definitely want to look elsewhere.
Overall, there's no standout reason to upgrade from previous editions. But if you're new to backside-blistering fun on four wheels, Fury 3 is worth a blast. [PSW]
Game InformerThis game controls like junk. [June 2005, p.136]
Play MagazineThe shoulder button helps cornering by speeding up the rate at which the background turns, but ultimately these ATVs feel more like semi trucks. [June 2005, p.86]
There were times when this game aggravated me so much I wanted to throw down the controller, before I realized I was holding a $250 controller.
Essentially a collection of the same old stuff we've been playing on the PS2 for the last few years, Blazin' Trails fails to tantalize the senses but rather frays the nerves with a sloppy frame rate and awkward controls.
Official U.S. Playstation MagazineA huge, solid game that's seriously crippled by an unworkable control scheme. [July 2005, p.78]
Hopefully if Climax releases another PSP version, they work the kinks out, because this one needs some polish.
It's not flawless in its execution, but I'd have to say there is just enough play in ATV Offroad Fury to keep fans happy.
Blazin' Trails needs more user-friendly control and a graphical overhaul--we're talking almost original PlayStation visuals here--but at the end of the day it's still a really enjoyable game.
There is plenty of content in the game, but the difficulty level and average controls may mean that most people don't see all that the game has to offer.
Unless you are a die-hard ATV fan who can put up with frustrating gameplay mechanics, I cannot recommend ATV Offroad Fury: Blazin' Trails to you.
The game is packed with modes, options and replayability, though it has a few annoying problems which takes away from the potential enjoyability of it all.
Because the PSP buttons (including the shoulder buttons) are in such close proximity to each other, your fingers are constantly tumbling over each other to pull off the right stunt.
Cheat Code CentralIt's loaded with modes and is a relatively deep game for a racer. You can still do a lot of tricks with it but overall the racing experience is hampered by its mechanical shortcomings.
Your competition is dead serious, the hazards are all fatal, and your only resource is the X button. While Blazin' Trails manages to cover a lot of different terrain, its shoddy gameplay prevents it from handling any of it well.
Awards & Rankings
#43 Most Discussed PSP Game of 2005
User Score
Generally favorable reviews- based on 16 Ratings
User score distribution:
Positive: 8 out of 9
Mixed: 0 out of 9
Negative: 1 out of 9
JoeR.Jun 18, 2005Tightest ATV game EVER, ya herd.
BillyF.May 15, 2005This game owns, online is great!
M.WebbMay 14, 2005The negative reviews from major sites for this game leave me scratching my head. The framerate is great! Yes, it slows to a crawl during a The negative reviews from major sites for this game leave me scratching my head. The framerate is great! Yes, it slows to a crawl during a racing wipeout animation but that only lasts for less than two seconds. Otherwise, the framerate during racing, including cornering, is quite solid. And what the negative reviews do not tell you, is that during Free Run, Hot Laps and Freestyle, when you are the only rider, the framerate goes through the roof and the driving as well as the wipeouts are thirty frames a second or better. The load times are on par with THPS Remix. Overall, Blazin' Trails is challenging to be sure with very realistic physics, but that is what makes it satisfying to play. The depth is astounding. There are tuning and customization options, many tracks, lots of ATV's and gear and a fantastic handlebar cam that looks identical to the one used in ATV Off-Road Fury 2 for the PS2. I have Metal Gear Acid, THPS Remix and Wipeout Pure and this game is superior to all of them. If you like racing games with realism and depth then you won't be dissapointed with Blazin' Trails.… Full Review »


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Record companies investigated over digital music pricing
The four major record companies are being investigated by New York attorney general Eliot Spitzer over whether they have violated antitrust laws in the pricing of songs sold by digital music services such as the iTunes Music Store.
"Spitzer's office recently began serving subpoenas on the major record companies—the Universal Music Group, a unit of Vivendi Universal; Sony BMG Music Entertainment, a joint venture of Sony and Bertelsmann; the EMI Group; and the Warner Music Group, according to people involved," reports The New York Times. "Some major record companies have been pushing to introduce flexible pricing, charging more for hit songs and less for oldies, for example. But some executives say that a more complex price structure would turn off buyers and hurt industry efforts to woo music listeners away from free, unauthorized file-swapping networks."
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These authors contributed equally to this work. [Correction added on 30 August 2012, after first online publication: An equal contribution statement and a second correspondence email have been added.]
Original Article
GAP161 targets and downregulates G3BP to suppress cell growth and potentiate cisplaitin-mediated cytotoxicity to colon carcinoma HCT116 cells
Version of Record online: 26 JUL 2012
DOI: 10.1111/j.1349-7006.2012.02361.x
© 2012 Japanese Cancer Association
Cancer Science
Volume 103, Issue 10, pages 1848–1856, October 2012
Additional Information
How to Cite
Zhang, H., Zhang, S., He, H., Zhao, W., Chen, J. and Shao, R.-g. (2012), GAP161 targets and downregulates G3BP to suppress cell growth and potentiate cisplaitin-mediated cytotoxicity to colon carcinoma HCT116 cells. Cancer Science, 103: 1848–1856. doi: 10.1111/j.1349-7006.2012.02361.x
Publication History
- Issue online: 8 OCT 2012
- Version of Record online: 26 JUL 2012
- Accepted manuscript online: 16 JUN 2012 02:00AM EST
- Manuscript Accepted: 11 JUN 2012
- Manuscript Revised: 7 JUN 2012
- Manuscript Received: 6 APR 2012
Funded by
- National Basic Research Program of China. Grant Number: 2009CB521807
- the National S&T Major Special Project on Major New Drug Innovation . Grant Number: 2009ZX09301-003
- Abstract
- Article
- References
- Cited By
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgments
- Disclosure Statement
- References
Ras-GTPase-activating protein SH3 domain-binding proteins (G3BP) are overexpressed in various human tumors and participate in several signaling pathways involved in growth, differentiation and apoptosis. G3BP interact with RasGAP (Ras-GTPase activating protein) only in growing cells and depend on Ras activation, and participate in the Ras signal pathway. Therefore, the blockage and downregulation of G3BP may be a new strategy for cancer therapy. In this report, we demonstrate that a novel peptide GAP161 blocked the functions of G3BP and markedly suppressed HCT116 cell growth through the induction of apoptosis. The peptide bound with G3BP, which interfered with the interaction of G3BP1 with RasGAP and further suppressed Ras signaling pathways. GAP161 downregulated G3BP1 and G3BP2 proteins. Similarly, the knockdown of G3BP substantially decreased the proliferation of HCT116 cells and inhibited Ras signal pathways. Furthermore, the downregulation of G3BP could enhance cisplatin-induced apoptosis and growth inhibition of HCT116 cells. We also found that GAP161 suppressed the growth of BALB/c mice bearing colon CT26 tumors and nude mice bearing HCT116 xenografts. These results suggest that downregulation of G3BP might be useful in cancer therapy and that GAP161 is a promising new therapeutic agent for cancers. (Cancer Sci, doi: 10.1111/j.1349-7006.2012.02361.x, 2012)
The G3BP protein family contains G3BP1 and G3BP2, with similar molecular structures. Both proteins are dramatically overexpressed in human cancers, especially in breast, head and neck, colon and lung.[1-3] G3BP are involved in a variety of growth-related signaling pathways that are involved in carcinogenesis and metastasis,[4, 5] including NF-κB, Ras signaling and the ubiquitin proteasome system.[1, 6-8] The expression level of G3BP1 changes reciprocally to the level of phosphatase and tensin homolog deleted on chromosome ten (PTEN). The negative effect of PTEN expression on the protein level of G3BP1 is recapitulated by direct inhibition of PI3K.[9] G3BP1 and G3BP2 also interact with p53 and MDM2 to modulate their expression and localization.[10] In addition, G3BP1 is an important marker of stress granules (SG). The NTF2-like domain of G3BP1 mediates its recruitment to SG influenced by Ras.[11] Stresses like hypoxia, heat shock and chemotherapy could induce SG formation, and SG could inhibit apoptosis by suppressing stress-responsive MAPK pathways.[12] Taken together, several lines of evidence suggest that the overexpressed G3BP in a wide range of cancers could be a target for anticancer therapy.
It is believed that RasGAP, a regulator of Ras and Rho GTP-binding proteins,[13, 14] have dual functions: the negative effect of GTPase pathways and downstream signaling transduction using effectors such as Raf-1, PI3K and MAPK.[15] The SH3 domain of RasGAP is contributed to binding of G3BP and is essential for Ras downstream signaling. Two independent groups found that p120-RasGAP was cleaved by caspases, which induced both anti-apoptotic and pro-apoptotic signals, depending on the extent of its cleavage by caspases.[16, 17] Cui et al.[18] report that two peptides derived from RasGAP SH3 domain were cytotoxicitic to tumor cells without significant effects on normal cells. The peptides also showed an obvious sensitizing effect to cisplatin (CDDP). CDDP could activate multiple intracellular pathways, and CDDP-induced NF-κB activation contributed to the resistance of cancer cells to cisplatin.[19-22] During NF-κB activation, phosphorylation of p65 subunit on Ser 536 is required for NF-κB to be fully activated.[23, 24]
In this study, we report a new peptide called GAP161, which blocked the cellular functions of G3BP, and triggered HCT116 cells to undergo apoptosis and potentiated the cytotoxicity of CDDP. Several G3BP-related molecules of transduction signaling pathways were detected that include Ras, ERK, NF-κB and Akt pathways. Meanwhile, we found that downregulation of G3BP inhibited HCT116 cell growth by inducing apoptosis similar to the effects of GAP161. We conclude that GAP161 can selectively target G3BP and promote apoptosis. Similar effects of GAP161 on HCT116 cells were also observed in mice bearing xenograft tumors.
Materials and Methods
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgments
- Disclosure Statement
- References
Peptide synthesis
GAP161, a cell-permeable peptide GAP161 was synthesized at Wuhan KatyGen Pharmaceuticals (Hubei, China). The molecular weight of GAP161 was 3811.6 Da. The peptides were dissolved in deionized water at a final concentration of 10 mM and stored at −20°C until further use.
Cell culture and reagents
Human colon carcinoma HCT116 cells were purchased from ATCC (Manassas, VA, USA). Cells were grown in DMEM (Gibco, Grand Island, NE, USA) supplemented with 10% FBS, 2 mM l-glutamine and 1% penicillin–streptomycin at 37°C in the presence of 5% CO2. Cisplatin (Sigma-Aldrich, St Louis, MO, USA) was diluted in PBS at a final concentration of 20 mM and stored at −20°C. U0126 and LY294002 were from Cell Signaling Technology (Beverly, MA, USA) and diluted in DMSO.
Confocal microscopy
Cells were grown on glass coverslips and incubated at 37°C for 24 h. Cells were fixed and permeabilized by ice-cold ethanol and then blocked with 1% BSA at room temperature. Coverslips were stained with the anti-G3BP1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by anti-mouse rhodamine–conjugated secondary antibody, and examined by Zeiss LSM710 confocal microscopy (Zeiss, Oberkochen, Germany).
Cell proliferation and cell cytotoxicity assay
Cell proliferation was determined by trypan blue dye exclusion assay. Cytotoxicity was determined by sulforhodamine B assay, as described previously.[25] For colony formation, cells were harvested after indicated treatment and seeded into six-well plates at a density of 1000 cells per well. Cells were fed with new culture medium every 4 days and colonies were counted and photographed after 9 days.
siRNA transfection
The sequences used for siRNA were synthesized by Ribobio Technology (Guangzhou, China). The siRNA sequence targeting G3BP1 was 5′-GCA ACA GUA UUU CGG UAU AdT dT-3′, and the target sequence for G3BP2 was 5′-GUA AGA AAG UAG CCA UAA AdT dT-3′. The control mock siRNA sequence was 5′-UUC UCC GAA CGU GUC ACG UdT dT-3′. Cells at 40–60% confluence in six-well culture dishes were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions.
Annexin V-FITC/propidium iodide staining and terminal deoxyribonucleotide transferase-mediated dUTP nick end-labeling assays
To quantify apoptosis, cells were stained with Annexin V and PI using an Annexin V-FITC/PI Apoptosis Kit (Roche, Mannheim, Germany), following the protocol provided by the manufacturer. For TUNEL assay, the tissues were treated as indicated and TUNEL staining was performed in situ using the DeadEnd fluorometric TUNEL system kit (Promega, Madison, WI, USA), according to the manufacturer's protocol. The images were captured by an image analysis system (Eclipse TE2000-U, Nikon, Japan).
Quantification of mRNAs using RT-qPCR
Total RNA was isolated from the cells and cDNA was generated by RT-PCR using a cDNA synthesis kit (TaKaRa, Dalian, China). The quantitative real-time PCR reactions were performed using 2× SYBR Premix Ex Taq II (TaKaRa). All samples were processed and measured using the CFX96TM Real-Time PCR Detection System (Bio-rad, Hercules, CA, USA). The following forward (F) and reverse (R) primers were used to amplify G3BP1, G3BP2 and GAPDH cDNAs: F-G3BP1: 5′-GAGAAGCCTAGTCCCCTGCT-3′, R-G3BP1: 5′-CCATTTGAATCCAATCCCCCA-3′; F-G3BP2: 5′-TTCAGTGACCAGTAAAAACCTGC-3′, R-G3BP2: 5′-GTGCTTTAACATGGGGTGGAA-3′ and F-GAPDH 5′-CATGAGAAGTATGACAACAGCCT-3′, R-GAPDH: 5′-AGTCCTTCCACGATACCAAAGT-3′. Relative expression levels of G3BP mRNA were determined using the comparative ΔΔC t method, with GAPDH as a control and relative to the not-transfected or not-treated cells.
Caspase-3/7 activity assay
After 48 h treatment, cells were subjected to caspase-3/7 activity measurement using the Caspase-Glo assay kit (Promega), according to the manufacturer's protocol. Caspase activities were expressed as increased fold compared to untreated cells.
Ras activation assay
The active form of Ras in cells was analyzed using a Ras activation assay kit according to the manufacturer's protocol (Upstate Biotechnology, Lake Placid, NY, USA). In brief, the treated cells were disrupted with Mg2+ lysis/wash buffer (MLB), and then the lysate was incubated at 4°C for 60 min, with beads coated with the GST fusion protein, Raf-1 Ras-binding domain, which specifically binds to Ras-GTP. The beads were then washed with MLB thrice, and the washed beads were suspended in 2× Laemmli sample buffer. SDS-PAGE and an immunoblot assay using anti-Ras were used to detect the relative amount of Ras-GTP.
Western blot and coimmunoprecipitation analysis
Whole cell lysates were used for immunoblot, as previously described.[26] For co-immunoprecipitation assay, after desired treatment, cells were collected and lysed in RIPA lysis buffer. Cell lysates were incubated at 4°C overnight with anti-G3BP1 antibodies, anti-G3BP2 antibodies or anti-RasGAP antibodies, followed by the incubation of protein A/G plus Agarose (Santa Cruz Biotechnology) for 6 h to overnight at 4°C. The beads were then washed with cold lysis buffer followed by centrifugation at 3000 g at 4°C for 5 min. The antibody binding proteins were resolved by SDS-PAGE and analyzed by western blot.
Primary antibodies were as follows: anti-caspase-3, anti-caspase-9, anti-cleaved caspase-7, anti-PARP, anti-phospho-MEK, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt, anti-Akt, anti-NF-κB p65, anti-phospho-NF-κB p65(Ser536) (Cell Signaling Technology), anti-G3BP1, anti-Bcl-2 and anti-RasGAP (Santa Cruz Biotechnology), anti-G3BP2 (Abcam) and anti-β-actin (Sigma).
GST pull-down assay
For binding assays, GST -G3BP proteins were preincubated with 30 μM GAP161 before HCT116 cell lysates were added. Client proteins associated with GST-G3BP were captured by glutathione Sepharose beads (Pierce, Rockford, IL, USA), while unbound proteins were removed by wash buffer. The fraction that was bound to the beads was analyzed by SDS-PAGE followed by immunoblotting with antibodies specific to G3BP1, G3BP2 and RasGAP.
In vivo tumor mouse model
For the mouse tumor model, female BALB/c mice were injected with 1.5 million mouse colon carcinoma CT26 cells subcutaneously on the right flank. The following day and thereafter daily for GAP161 and every other day for CDDP, the mice were injected intraperitoneally with PBS, 25, 50 and 100 mg/kg of GAP161, 1 mg/kg CDDP, or a combination of GAP161 and CDDP. After 11 days, all mice were weighed and killed, and the tumors were excised. Tumors were weighed, and the mean tumor weight was calculated.
For the HCT116 xenograft tumor model, female BALB/c nude mice (18–22 g) were implanted by subcutaneous injection of 5 × 106 cells on the right flank. After 3 weeks, tumors were aseptically dissected and pieces of tumor tissue (2 mm3 in size) were transplanted subcutaneously by trocars into mice. When tumor size was over 100 mm3, mice were divided into groups (n = 6) and injected intraperitoneally with PBS and GAP161 each day, or 0.5 mg/kg CDDP every other day, or a combination of these. Tumor size was measured and tumor volume was determined by length × width2/2. At day 24, tumors were taken from mice and weighed. The tumor growth inhibition (TGI) for each group was calculated as 1 – tumor weight (treatment)/tumor weight (control) × 100%.
For histologic examination, the final tumors were cut into two. Half was for western blot analysis; the other half was formalin-fixed and embedded in paraffin blocks and then cut into thin sections. The tissue sections were either immunostained with antibody against G3BP and NF-κB. Stained sections were observed using a Leica microscope (×400; Leica, Wetzlar, Germany).
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgments
- Disclosure Statement
- References
GAP161 suppresses HCT116 cell growth and induces apoptosis
Based on the structural model of G3BP-RasGAP complex built by Cui et al.[18] we designed a peptide by simulation of its interaction with G3BP. To increase cell permeability, the designed peptide was fused with a short peptide derived from the human immunodeficiency virus TAT protein (HIV48–57). The amino acid sequence of the final designed peptide is shown in (Fig. 1a) and is named GAP161. To determine the potential effect on cell growth, HCT116 cells were treated with increasing concentrations of GAP161 for various periods of time. As shown in the left panel of Figure 1(b), GAP161 inhibited the proliferation of HCT116 cells in a dose-dependent and a time-dependent manner. The long-term cell viability was measured by colony formation assay (Fig. 1b, right panel), which also showed the cytotoxicity in a dose-dependent manner. FACS analysis showed that GAP161 markedly increased accumulation of cells in the sub-G1 phase (Fig. 1c), indicating that GAP161 suppressed cell growth by promoting apoptosis. To test this hypothesis, Annexin V/PI staining was performed and the results showed that GAP161 significantly induced apoptosis in HCT116 cells (Fig. 1c, lower panel). As shown in Figure 1(d), GAP161 markedly induced the cleavage of caspase-3 and 7 and downregulated the expression of Bcl-2 protein. Together, these results showed that GAP161 could inhibit the growth of cultured HCT116 cells by activating the apoptotic signal pathway.
GAP161 binds and downregulates G3BP in HCT116 cells
To assess the cellular uptake and distribution of GAP161, the peptide was labeled with FITC and incubated with HCT116 cells for various periods of time. As shown in Figure 2(a), GAP161 began to localize on the cell membrane after only 15 min incubation. After 30 min incubation, GAP161 entered cells and mostly located in the cytoplasm. When cells were incubated for 24 h, some of the peptides were distributed into the nucleus. To see whether the distribution of GAP161 was correlated with G3BP, cells were treated with the FITC-labeled GAP161 for 60 min and then fixed. The fixed cells were incubated with anti-G3BP antibody followed by an incubation with rhodamine (red)-conjugated secondary antibody for monitoring G3BP. The stained cells were examined under a confocal microscopy. We found that GAP161 and G3BP were co-localized mainly in the cytoplasm (Fig. 2b). To investigate whether GAP161 directly interacted with G3BP, immunoprecipitation (anti-G3BP antibody), western blot and Typhoon scanning were used to analyze FITC-GAP161-treated HCT116 cells (Fig. 2c). Consistent with their co-localization inside the cells, the immunoprecipitation assay showed that GAP161 directly interacted with G3BP.
After confirming the interaction between GAP161 and G3BP, we investigated whether GAP161 affected the interaction of G3BP with RasGAP. As shown in Figure 2(d), RasGAP interacted with G3BP in HCT116 cells and a reciprocal co-IP experiment indicated that GAP161 decreased the binding of RasGAP to G3BP. GAP161 did not affect the level of RasGAP protein, but downregulated the level of G3BP1 and G3BP2 (Fig. 2d). Moreover, a GST fusion G3BP affinity chromatography assay was used to examine the ability of GAP161 to disrupt the G3BP association with RasGAP. The addition of GAP161 to the cell lysates led to the release of RasGAP. GAP161 appeared to decrease the binding of RasGAP to G3BP (Fig. 2d). Western blot assay showed that GAP161 significantly decreased G3BP level in a dose-dependent and a time-dependent manner (Fig. 2e). However, the level of G3BP mRNA did not change after the treatment, indicating that the decrease of G3BP protein was not due to transcription (Fig. 2f).
Knockdown of G3BP inhibits HCT116 cell growth through induction of apoptosis
The results described above suggest that GAP161-induced apoptosis was caused by the downregulation of G3BP proteins. To test this possibility, we designed specific siRNA against G3BP1 and G3BP2. RT-qPCR and western blotting analyses showed that transcription and expression of G3BP in HCT116 cells were dramatically downregulated by siRNA (Fig. 3a). Notably, the knockdown of G3BP significantly inhibited HCT116 cell growth and colony formation (Fig. 3b). Furthermore, Annexin V staining revealed that G3BP siRNA induced HCT116 cells to undergo apoptosis (Fig. 3c). Meanwhile, apoptosis-related Bcl-2 protein was downregulated and the active cleavages of caspasse-3, 9 and PARP were upregulated (Fig. 3d).
Downregulation of G3BP impaires Ras signaling pathway
Ras is a small GTPase responsible for transducing molecular signals. The function of G3BP is important in the oncogenic Ras signaling pathway.[3] We found that GAP161 could decrease the abundance of RasGTP, but did not affect the cellular level of Ras protein (Fig. 4a). The key signal pathway from activated Ras is the ERK/MAPK cascade and PI3K pathway. Western blot analyses showed that GAP161 clearly inhibited MEK, ERK activity and Akt phosphorylation, but had no significant effect on total ERK and Akt protein levels (Fig. 4b). It has been reported that NF-κB activity could be modulated by ERK and Akt.[27] Interestingly, we also found that GAP161 significantly downregulated the phosphorylation and expression of NF-κB.
To confirm these results, we monitored Ras activity in G3BP-silenced HCT116 cells. We found that knockdown of G3BP deceased the level of RasGTP and impaired Ras activity (Fig. 4d). Consequently, the activities of downstream signaling proteins, such as MEK/ERK, Akt and NF-κB, were downregulated. In addition, the MEK and PI3K inhibitors U0126 and LY294002 did not affect the cellular level of G3BP proteins, indicating that G3BP were upstream of MEK and Akt (Fig. 4c).
Downregulation of G3BP sensitizes HCT116 cells to CDDP
It has been reported that concurrent blockade of the NF-κB and Akt pathways potently sensitizes cancer cells to chemotherapeutics.[19, 28] We found that a combination of GAP161 and CDDP significantly inhibited the growth and colony formation of HCT116 cells as compared with the treatment of CDDP or GAP161 alone (Fig. 5a). Thus, GAP161 synergistically enhanced CDDP-induced cytotoxicity in HCT116 cells. The knockdown of G3BP by siRNA also significantly sensitized HCT116 cells to CDDP (Fig. 5b).
Consistent with the cell viability results, GAP161 markedly potentiated CDDP-induced sub-G1 phase accumulation and Annexin V–positive cells (Fig. 5c). Western blotting analysis showed that CDDP modestly induced activations of caspase-3 and caspase-7 (Fig. 5d). However, GAP161 markedly enhanced CDDP-induced cleavages of caspase-3 and caspase-7 (Fig. 5d). These results were confirmed by caspase-3 and caspase-7 activity assays (Fig. 5e). Bcl-2 was also downregulated in the combination-treated cells compared with GAP161 treatment (Fig. 5d). These results further confirmed that the synergetic effect of GAP161 and CDDP was mediated through activation of apoptotic signal pathway. Meanwhile, GAP161 downregulated the level of RasGTP and inhibited the activation of Ras (Fig. 5f). CDDP-induced activation of ERK and NF-κB played an important role in chemoresistance.[20, 22] As shown in Figure 5(f), CDDP induced ERK phosphorylation and aberrantly activated NF-κB, but had no obvious effect on Akt activity. In agreement with GAP161 treatment, the combination of CDDP and GAP161 clearly inhibited the activation of ERK and Akt, and significantly downregulated the phosphorylation and expression of NF-κB.
GAP161 suppress tumor growth and sensitized CDDP in vivo
We next conducted an in vivo experiment to examine the potential effect of GAP161 on tumor growth. HCT116 xenograft tumors (50–100 mm3) were treated intraperitoneally with various concentrations of GAP161 once per day. As shown in Figure 6(a), GAP161 significantly reduced tumor volume in a dose-dependent and time-dependent manner. On day 24, the average tumor weight in the GAP161-treated group is lower than that in the PBS group (Fig. 6b).
To investigate the effects of GAP161 on tumor growth in vivo when combined with CDDP, we conducted two types of experiments to examine the anti-tumor effect of GAP161 and the synergistic effect to CDDP, particularly under the doses of CDDP with poor or marginal effects. In the first experiment, female BALB/c mice were injected subcutaneously with 1.5 million mouse colon carcinoma CT26 cells on the right flank. The following day and thereafter daily for GAP161 and every other day for CDDP, the mice were injected intraperitoneally with PBS, GAP161, CDDP alone, or a combination of GAP161 and CDDP. A combination of CDDP with GAP161 displayed markedly antitumor effects (Table 1).
|Dosage (mg/kg)
|No. of mice (begin/end)
|Body weight change (g)
|Tumor weight (g) (mean ± SD)
|2.47 ± 0.31
|1.53 ± 0.39*
|2.27 ± 0.42
|1.91 ± 0.43*
|20 + 1
|0.97 ± 0.32*,**
|40 + 1
|0.81 ± 0.25*,**
In the second experiment, we transplanted HCT116 cells into nude mice and treated them with GAP161and CDDP. As shown in Figure 6(c), GAP161 could inhibit the growth of HCT116 tumors and markedly enhanced CDDP-induced growth inhibition. On day 24, the tumor weight of GAP161-treated group is lower than that of the PBS group, and the tumor weights of the combination-treated groups were significantly lower than those of the CDDP group (Fig. 6d). Outward signs of drug toxicity were minimal, and there was no significant weight-loss in the treated groups (data not shown). According to the tumor weight, 0.5 mg/kg of CDDP alone showed a slight inhibitory effect, with 22.5% TGI as compared with the PBS control. In addition, 30 and 60 mg/kg of GAP161 showed modest therapeutic efficacy, with 17.1% and 31.5% TGI, respectively. The combination of GAP161 and CDDP displayed markedly antitumor effects, with 45.95% and 61.26% TGI (P < 0.05 vs CDDP), respectively.
Moreover, GAP161 reduced the expression of G3BP and inhibited the activation of ERK, Akt and NF-κB, and promoted the cleavage of caspase-3 in HCT116 xenograft tumors (Fig. 7a). Inconsistent with the in vitro effect, GAP161 decreased the binding of RasGAP to G3BP in vivo (Fig. 7b). H&E staining and TUNEL staining revealed large fractions of apoptotic cells in the tumors treated with GAP161 or the combination of GAP161 and CDDP, but not in those treated with CDDP or PBS (Fig. 7c). In addition, immunohistochemistry assay showed that G3BP and NF-κB were downregulated in GAP161-treated tumors (Fig. 7c). These data showed that GAP161 could effectively inhibit the tumor growth in vivo through induction of apoptosis.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgments
- Disclosure Statement
- References
G3BP were first isolated in a screen for proteins that bind to SH3 domain of RasGAP,[29] and the NTF2-like domain was responsible for its binding to the SH3 domain of RasGAP.[30] Parker et al.[31] developed a monoclonal antibody directed against the NTF2-like domain of G3BP that showed a specific effect in the therapy for the G3BP-related tumors. According to the G3BP NTF2-like domain and RasGAP SH3 domain interaction model established by Cui et al.[18] we designed a new peptide GAP161 through amino acid substitution, which, theoretically, had strong interaction with G3BP. To improve the penetrating ability, we therefore synthesized GAP161 covalently bound to the amino acids carrier peptide HIV-TAT48–57. Glycine residues were inserted between the TAT and GAP161 as spacers to allow flexibility. GAP161directly interacted with G3BP proteins, blocked their binding to RasGAP, and downregulated the cellular level of G3BP proteins. Consistent with this, GAP161 impaired Ras activity, strongly inhibited HCT116 cell growth, and significantly sensitized HCT116 cells to CDDP both in vitro and in vivo.
RasGAP is an important protein in the modulation of Ras signal transduction. RasGAP and G3BP interaction occurs only in growing cells and both proteins are recruited to activate Ras,[6, 29] suggesting that G3BP could participate in Ras signal pathway through RasGAP. In this study, the binding of RasGAP to G3BP was decreased in GAP161-treated cells, demonstrating that GAP161 competed with RasGAP to bind G3BP. At the same time, the level of RasGTP in GAP161-treated cells was much lower than that of the control and the CDDP group. GAP161 competitively bound to G3BP and released RasGAP to inactivate Ras from its active GTP-bound form to its inactive GDP-bound form by enhancing the endogenous GTPase activity of Ras.
In consistent with the inactivation of Ras in GAP161-treated cells, the downstream targets of Ras signaling were inhibited, including MEK, ERK and Akt. In contrast, CDDP abnormally induced ERK phosphorylation, which may contribute to antagonizing apoptosis.[20, 22] Previously, we have demonstrated that MEK inhibitor U0126 and PI3K inhibitor LY294002 could sensitize HCT116 cells to CDDP.[32] When HCT116 cells were treated with GAP161 plus CDDP, CDDP-induced ERK activation was blocked. In addition, NF-κB was significantly downregulated in GAP161-treated cells. Taken together, the multiple-target inhibitions by GAP161 might contribute to the synergism.
Overexpressed G3BP induce numerous downstream signaling events, including the activation of Ras signaling, MAPK activatio and phosphorylation of multiple signaling molecules.[6, 33, 34] Interestingly, GAP161 significantly decreased G3BP protein levels. This effect was probably not caused by influencing the stability of the G3BP transcription because there was no significant change in the steady-state levels of G3BP mRNA. G3BP could participate in ubiquitin proteasome system (USP10) signaling pathways but G3BP itself was not a substrate of USP10.[8] Similarly, proteasome inhibitor MG132 could not reverse GAP161-induced downregulation of G3BP (data not shown). So far, whether GAP161 specifically represses the post-transcription or initiates the degradation of G3BP remains unknown. Therefore, it is important to carry out further investigations to identify the detailed regulatory network.
To mimic GAP161-induced downregulation of G3BP, the knockdown of G3BP by RNA interfering also suppressed HCT116 cell growth and Ras signaling molecules (Fig. 4d). Furthermore, the knockdown of G3BP1 modestly decreased NF-κB, while the knockdown of G3BP2 significantly downregulated NF-κB. Moreover, MEK inhibitor U0126 and PI3K inhibitor LY294002 did not affect G3BP protein levels in HCT116 cells, indicating that G3BP was upstream of MEK and Akt. These data suggested that not only G3BP1 but also G3BP2 played an important role in cell proliferation and that GAP161-induced downregulation of G3BP inhibited HCT116 cell growth and sensitized HCT116 cells to CDDP.
In summary, GAP161 targeting to the NTF2 domain of G3BP showed antitumor activity and potentiated CDDP-induced cytotoxicity of the HCT116 cells both in vitro and in vivo. GAP161 blocked RasGAP binding to G3BP and downregulated G3BP protein to inhibit cell growth and sensitize HCT116 cells to CDDP. The knockdown of G3BP also suppressed HCT116 cell growth and potentiated CDDP-induced cell death. These findings suggest that downregulation of G3BP1 and G3BP2 is a promising strategy for cancer treatment.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgments
- Disclosure Statement
- References
We thank Yongjie Xu (Wright State University School of Medicine) for helpful discussion and suggestions. This research was supported by grants from the National Basic Research Program of China (No. 2009CB521807) and the National S&T Major Special Project on Major New Drug Innovation (No. 2009ZX09301-003).
Disclosure Statement
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgments
- Disclosure Statement
- References
The authors declare no conflict of interest.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgments
- Disclosure Statement
- References
- 1Heregulin induces expression, ATPase activity, and nuclear localization of G3BP, a Ras signaling component, in human breast tumors. Cancer Res 2002; 62: 1251–5., , , , , .
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- 3G3BP is overexpressed in human tumors and promotes S phase entry. Cancer Lett 2001; 162: 213–21., , , , .
- 4Intracellular CD24 inhibits cell invasion by posttranscriptional regulation of BART through interaction with G3BP. Cancer Res 2011; 71: 895–905., , .
- 5The N-terminal domain of G3BP enhances cell motility and invasion by posttranscriptional regulation of BART. Mol Cancer Res 2011; 9: 856–66., , .
- 6A novel phosphorylation-dependent RNase activity of GAP-SH3 binding protein: a potential link between signal transduction and RNA stability. Mol Cell Biol 1998; 18: 3956–65., , et al.
- 7IkappaBalpha and IkappaBalpha/NF-kappa B complexes are retained in the cytoplasm through interaction with a novel partner, RasGAP SH3-binding protein 2. J Biol Chem 2000; 275: 36441–9., , , .
- 8Ras-GAP SH3 domain binding protein (G3BP) is a modulator of USP10, a novel human ubiquitin specific protease. Oncogene 2001; 20: 3869–79., , .
- 9Modulation of specific protein expression levels by PTEN: identification of AKAP121, DHFR, G3BP, Rap1, and RCC1 as potential targets of PTEN. Oncogene 2005; 24: 3819–29., , , , , .
- 10Modulation of p53 and MDM2 activity by novel interaction with Ras-GAP binding proteins (G3BP). Oncogene 2007; 26: 4209–15., , , , .
- 11The RasGAP-associated endoribonuclease G3BP assembles stress granules. J Cell Biol 2003; 160: 823–31., , et al.
- 12Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways. Nat Cell Biol 2008; 10: 1324–32., , , , .
- 13Increasing complexity of Ras signaling. Oncogene 1998; 17: 1395–413., , , , .
- 14Ras-GAP controls Rho-mediated cytoskeletal reorganization through its SH3 domain. Mol Cell Biol 1998; 18: 5567–78., , .
- 15GEFs and GAPs: critical elements in the control of small G proteins. Cell 2007; 129: 865–77., , .
- 16A RasGAP-derived cell permeable peptide potently enhances genotoxin-induced cytotoxicity in tumor cells. Oncogene 2004; 23: 8971–8., , , , .
- 17Antiapoptotic signaling generated by caspase-induced cleavage of RasGAP. Mol Cell Biol 2001; 21: 5346–58., .
- 18Structure-based design of peptides against G3BP with cytotoxicity on tumor cells. J Chem Inf Model 2010; 50: 380–7., , et al.
- 19Blockage of NF-kappaB by IKKbeta- or RelA-siRNA rather than the NF-kappaB super-suppressor IkappaBalpha mutant potentiates adriamycin-induced cytotoxicity in lung cancer cells. J Cell Biochem 2008; 105: 554–61., , , , , .
- 20Cisplatin-induced activation of mitogen-activated protein kinases in ovarian carcinoma cells: inhibition of extracellular signal-regulated kinase activity increases sensitivity to cisplatin. Clin Cancer Res 1999; 5: 1007–14., , , .
- 21Biological and chemical inhibitors of NF-kappaB sensitize SiHa cells to cisplatin-induced apoptosis. Mol Carcinog 2005; 44: 51–9., , , , .
- 22Acquisition of chemoresistance in intrahepatic cholangiocarcinoma cells by activation of AKT and extracellular signal-regulated kinase (ERK)1/2. Biochem Biophys Res Commun 2011; 405: 333–7., , , , .
- 23NF-kappa B p65 transactivation domain is involved in the NF-kappa B-inducing kinase pathway. Biochem Biophys Res Commun 2003; 301: 583–90., , , , , .
- 24Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation. Trends Biochem Sci 2005; 30: 43–52., , , .
- 25Suppression of N-Ras by shRNA-expressing plasmid increases sensitivity of HepG2 cells to vincristine-induced growth inhibition. Cancer Gene Ther 2009; 16: 693–702., , et al.
- 26MR-1 modulates proliferation and migration of human hepatoma HepG2 cells through myosin light chains-2 (MLC2)/focal adhesion kinase (FAK)/Akt signaling pathway. J Biol Chem 2008; 283: 35598–605., , et al.
- 27NF-kB in development and progression of human cancer. Virchows Arch 2005; 446: 475–82., , , .
- 28Concurrent blockade of the NF-kappaB and Akt pathways potently sensitizes cancer cells to chemotherapeutic-induced cytotoxicity. Cancer Lett 2010; 295: 38–43., , et al.
- 29A Ras-GTPase-activating protein SH3-domain-binding protein. Mol Cell Biol 1996; 16: 2561–9., , et al.
- 30Characterization of G3BPs: tissue specific expression, chromosomal localisation and rasGAP(120) binding studies. J Cell Biochem 2001; 84: 173–87., , , , , .
- 31Monoclonal antibodies directed against the G3BP protein, and uses. US patent 7,001,980. 2006; 21 Feb., , , .
- 32RasGAP-derived peptide 38GAP potentiates the cytotoxicity of cisplatin through inhibitions of Akt, ERK and NF-kappaB in colon carcinoma HCT116 cells. Cancer Lett 2011; 308: 62–70., , et al.
- 33TNF-R1 signaling: a beautiful pathway. Science 2002; 296: 1634–5., .
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Political corruption is not unique to New Jersey. It is a malady as old as civilization itself. In an explosion of materialism, civilization offered the promise of more to agricultural villagers - trade and commerce (usually connected to war), growth, development, jobs. The rulers cashed in; the masses got hopes and "stuff."
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Kannel Not sending SMS
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With a major shift to technology in classrooms, one question Union School District administrators are being asked is whether textbooks still have a place in the classroom, and if so, to what extent?
The textbook discussion took place Monday night at the board meeting after several parents complained to board members that their child didn't have a textbook to bring home.
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Courtesy of Radical, CBR presents a preview of "After Dark" #0, the specially priced $1.00 introduction to the world created by Wesley Snipes and Antoine Fuqua, written by Peter Milligan with art by Jeff Nentrup, in stores June 30.
Creator puts "Battle Angel Alita: Last Order" on indefinite hiatus! Wisconsin store fire investigated as arson! New York police search for ... Catwoman! A look at Wonder Woman's anniversaries! Bad writing and art! Plus more!
Last week, CBR's reviewers looked at two dozen new comics, including the latest issues from "Zatanna," "Supergirl," "Secret Warriors," "American Vampire," "Green Lantern Corps," "Incorruptible" and much more.
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Marvel Studios has announced that Academy Award winner Tommy Lee Jones has joined the cast of "Captain America: The First Avenger," taking on the role of US Army Colonel Chester Phillips.
Timothy Callahan reviewed "The Amazing Spider-Man" #635 written by Joe Kelly, giving it 4.5 stars and saying, "[W]ith this "Grim Hunt" arc, the art is spectacular and the story is just as powerful."
Doug Zawissza gave "Power Girl" #13 a 4 star review, saying of Judd Winick and Sam Basri's first issue, "The best thing to do here was to just go in a different direction. So far, that plan is looking like a great idea."
Dynamite has provided CBR with advance looks at new comics on sale this Wednesday, June 30, including "Green Hornet: Parallel Lives" #1, "The Expendables" #3, "Prince of Persia: Before the Sandstorm" #1 and more.
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Sony execs have reportedly reviewed screen tests for the new Spider-Man and are expected to make their decision and subsequent casting announcement shortly. SPINOFF ONLINE has the details.
Jeph Loeb has been appointed Executive Vice President, Head of Television, a newly created division of Marvel Entertainment. In this position, Loeb will oversee the translation of Marvel characters to television.
BOOM! Studios has provided CBR with advance looks at new comics on sale June 30, including "Do Androids Dream of Electric Sheep?" #12, "DADOES: Dust to Dust" #2, "Muppet Show" #7, "Toy Story" #7, and more!
In the latest installment of this CSBG feature examining comics that comment on other comics, see what happened when a Hulk/Doc Ock fight in the pages of Spider-Man was later referenced in an issue of the Hulk's own title!
With the debut of the new season of "Futurama" marking the latest in a line of shows returning from the cancellation graveyard, SPINOFF asks: Which other canceled series deserves another chance?
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Hilton Belfast hotel, Belfast
- Free WiFi in lobby
Main amenities
- 198 guestrooms
- Restaurant and bar/lounge
- Breakfast available
- 24-hour fitness center
- Self parking
- Business center
- Limo/town car service
- 24-hour front desk
- Air conditioning
- Daily housekeeping
- Car rentals on site
- Front desk safe
For families
- Cribs/infant beds available
- Rollaway/extra beds available
- Connecting/adjoining rooms available
- Private bathroom
- Free toiletries
- Daily housekeeping
What's around
- In Belfast Central District
- Waterfront Hall (0 mi / 0.1 km)
- Belfast City Hall (0.5 mi / 0.8 km)
- Ulster Hall (0.6 mi / 0.9 km)
- Grand Opera House (0.6 mi / 1 km)
- Odyssey Arena (0.6 mi / 1 km)
Hilton Belfast hotel, Belfast
Hotel Info: 855-239-9397from $87
- Twin Hilton Executive Room
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- Hilton Deluxe, Twin Room, Non Smoking
- King Hilton Guest Room
- Twin Hilton Guest Room
- Family Room
- King Studio Room
- Hilton, Deluxe Room, 1 King Bed, Non Smoking
- Family Room
- Executive Suite
Also popular with travelers from the USA
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Key facts
Hotel size
- This hotel has 198 rooms
- This hotel is arranged over 12 floors
- Check-in time starts at 3 PM
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Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Pets allowed (dogs only)
- Up to 10 lb
Extra people
- Roll-away beds available, charges may apply*
- Free WiFi in public areas
- WiFi in rooms *
- Self parking *
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In the hotel
- Full breakfast daily (surcharge)
- Restaurant
- Bar/lounge
- 24-hour room service
- 24-hour fitness facilities
- Personal motorized watercraft on site
- Golfing nearby
- Hiking/biking trails nearby
- Water skiing nearby
- Business center
- Number of meeting rooms - 9
- Meeting rooms 10
- 24-hour front desk
- Concierge services
- Limo or Town Car service available
- Dry cleaning/laundry service
- Laundry facilities
- Free newspapers in lobby
- Luggage storage
- Wedding services
- Multilingual staff
- Elevator/lift
- Safe-deposit box at front desk
- Accessible bathroom
- In-room accessibility
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Minibar
- Coffee/tea maker
- Iron/ironing board (on request)
- Private bathroom
- Shower/tub combination
- Free toiletries
- Hair dryer
- Pay movies
- Cable TV channels
- Video-game console
- Desk
- Free weekday newspaper
- WiFi (surcharge)
- Phone
- Daily housekeeping
- In-room safe
- Connecting/adjoining rooms available
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Special features
DiningSonoma Restaurant - This restaurant serves dinner only. A children's menu is available. Reservations are required.
Cables Bar - This tapas bar serves dinner only.
Hilton Belfast hotel, Belfast's small print
Also known as
- Belfast Hilton
- Belfast Hilton International
- Hilton Belfast Hotel Belfast
- Hilton International Belfast
- Belfast Hilton hotel
- Belfast hotel
- Belfast hotel Hilton
- Hilton Belfast
- Hilton Belfast hotel
- Hilton hotel Belfast
- hotel Belfast
- hotel Hilton Belfast
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
Optional extras
Self parking costs GBP 18 per night
Parking is available nearby and costs GBP 18 per night
Full breakfast is offered for an extra charge of GBP 11 per person (approximately)
WiFi is available in guestrooms for GBP 14.99 per 24-hour period (rates may vary)
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Recent reviews
Excellent 4.2 /5 from 478 reviews
Center City is very nice. Parking was expensive.
The parking structure is extraordinarily narrow and difficult to park. I had prepaid for executive King, but checking in at 22:00, it had been taken. They put me in a room on 2nd floor instead of executive level. No big deal, the room was nice enough. No free wifi. I think you may be able to get free wifi if you sign up for Hilton's loyalty program, but I didn't try. I ordered a sandwich from room service 30 min before end of room service hours. The phone rang about 30 times before someone, who obviously didn't want to take any more orders, took my order. Great location for views and proximity to downtown. Had a great time at the St George's market Sunday morning, just two blocks from hotel. I will try a different hotel during my next trip to Belfast.
Walking distance from train station, city center and Titantic museum. Great location on the River Lagan.
The staff was excellent and went out of their way to be helpful by arranging meals, transportation and entertainment, storing bags and lending an umbrella. They even found an item I left behind and shipped it to me.
Wifi is a bit pricy, otherwise it was a great stay. Hotel restaurant is great BTW.
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