text:
A: No. General Admission tickets include all exhibitions on view. Tickets are not timed and do not sell out.

Can I host my wedding, bar/bat mitzvah, corporate meeting or dinner, birthday, graduation, or holiday party at the museum?

A: Yes, learn more on our Special Events page.

What are the benefits and eligibility surrounding the IDNYC Card Program?

A: The Museum of the City of New York is offering all IDNYC card holders a free Individual membership for one year. Please click here to learn more about the program. To redeem your membership, please visit the City Museum from 10:00 a.m. to 6:00 p.m. and have your valid IDNYC card with you.

Where can I read about the Museum’s Privacy Statement for this website?

A: Terms of Use can be found here.

Which Museum affiliates receive free admission?

A: Teachers/Educators, Columbia University and NYU students, employees of the City of New York, AAM (American Association of Museums) members, employees of other museums, and active military all receive free admission to the Museum (with valid I.D.).

What is the City Museum's I'm a "Neighbor" program?

A: If you live or work in zip codes 10029, 10035, or 10037 (the areas bounded by 96th & 142nd Streets to the south and north, respectively, and by Fifth Avenue and East and Harlem Rivers to the west and east, respectively), visit the Museum free of charge. Mention “I’m a neighbor,” and the suggested admission charge will be waived.

Are baby strollers or backpacks permitted in the Museum?

A: All bags are subject to inspection. Visitors may check these items in the self-check area on the first floor. You may be required to store oversized items or luggage; if so, the Museum’s staff will direct you to a separate storage area. Single strollers are permitted in the galleries subject to the discretion of gallery guards.

Is there a coatroom / coat check?

A: Due to construction, there is a temporary self-serve coat check on the first floor.

Is photography permitted in the Museum?

A: Still photography for personal, noncommercial use is permitted unless otherwise noted. Tripods and flash photography are not permitted.

What are the rules regarding use of mobile devices in the Museum?

A: Feel free to text, tweet, and share what you see during your visit! As a courtesy to other visitors, please set your phone to vibrate and take all voice calls outside of the galleries.

Is there WiFi in the Museum?

A: Free WiFi access is now available throughout the Museum's building as well as on the Front Terrance. Connect with your friends and family: share insights on our special exhibitions and your visit.

Are visitors permitted to sketch in the Museum?

A: Pencils, sketchbooks, and notebooks are permitted. However, pens, paints, and easels are not permitted.

Is there a place to eat in the Museum?

A: Chalsty’s Cafe is located on the 2nd floor and is open seven days a week, 10am–5pm.

Is the Museum accessible to wheelchairs and the disabled?

A: The City Museum strives to accommodate visitors of all abilities. Ramp access is available on 103rd Street between Fifth and Madison Avenues. All exhibition videos are open captioned. Sign language interpretation and specialized descriptive tours are available upon advanced request. Please email for more information.

What if my question isn’t answered here?

A: For further information about the Museum of the City of New York, contact Visitor Services at 212.534.1672, Mon–Fri, 9 am–5 pm, or e-mail your question to


Content-Type: application/http; msgtype=response

WARC-Type: response

text:
SingStar Beatles confirmed

Ben Parfitt
SingStar Beatles confirmed

A SingStar-branded PS3 SKU of EA’s The Beatles game, as exclusively revealed by MCV last month, has officially been confirmed for retail.

MTV itself moved to rubbish the claims just 24 hours after MCV broke the shock news, but now Kotaku has got the first pics of the upcoming PS3 SKU.

The Beatles: Rockband: SingStar includes a copy of the game and two wired PS3 microphones of the same sort that come with Sony’s own SingStar series. The included game is compatible with both the standard Rock Band drums and guitar, both of which are available separately.

The game will be released later this month, with Kotaku speculating that it will be a PAL only release.

Tags: Sony , PlayStation , ps3 , ea , rock , mtv , singstar , band , beatles

Follow us on

  • RSS

warcinfo:
robots: classic
hostname: ip-10-171-96-226.ec2.internal
software: Nutch 1.6 (CC)/CC WarcExport 1.0
isPartOf: CC-MAIN-2015-35
operator: CommonCrawl Admin
description: Wide crawl of the web for August 2015
publisher: CommonCrawl
format: WARC File Format 1.0
conformsTo: http://bibnum.bnf.fr/WARC/WARC_ISO_28500_version1_latestdraft.pdf


text:
MINNEAPOLIS, MN– JULY 28, 2009– Activision, Inc. (Nasdaq: ATVI) announced today that Cabela’s Outdoor Adventures will be released this fall on the Xbox 360® video game and entertainment system from Microsoft, PlayStation®2 and PLAYSTATION®3 computer entertainment systems, Wii™, and PC. Cabela’s Outdoor Adventures offers outdoor enthusiasts a simulation-style, seamlessly integrated experience that includes big game hunting, fishing and bird shooting.

“Never before has a Cabela's game had so much variety,” said David Oxford, Activision Publishing.“We went back to the drawing board with the folks at Cabela's and listened closely to the serious enthusiasts of each of these disciplines to make sure Cabela’s Outdoor Adventures is the most complete game ever for sportsmen.”

Cabela’s Outdoor Adventures features North America’s top outdoor destinations, over 50 hunting and fishing adventures and intensive use of gear and tactics, such as animal calls and stealth skills. Emphasis is placed on moments that are instinctively associated with hunting and fishing, like stalking the animals, squeezing the trigger, hooking a fish or snap-shooting a flushed bird, all while respecting real life hunting rules and regulations. Players can also upgrade their hunting gear with authentically branded real life equipment.

“I commend Activision for their commitment to simulating the outdoor sports in a challenging, fun and exciting format,” said Bryan Stave of Cabela's.“Cabela’s Outdoor Adventures gives real enthusiasts their fix when they can’t be in a forest or on a lake.”

Brand new to the franchise is an all-new shooting system known as VITALS (Visually Integrated Targeting and Lock-on System) that allows the player to focus on delivering an accurate shot to the prey’s vitals, while also creating a closer and more personal view of the animals as they react realistically to being hunted. Even better, you can create your trophy buck - customize your deer’s rack, body and color. For the new players, the game offers integrated tutorials and guided hunts.

For more information visit

Cabela’s Incorporated, headquartered in Sidney, Nebraska, is a leading specialty retailer, and the world’s largest direct marketer, of hunting, fishing, camping and related outdoor merchandise. Since the Company’s founding in 1961, Cabela’s has grown to become one of the most well-known outdoor recreation brands in the world, and has long been recognized as the World’s Foremost Outfitter. Through Cabela’s growing number of retail stores and its well-established direct business, it offers a wide and distinctive selection of high-quality outdoor products at competitive prices while providing superior customer service. Cabela’s also issues the Cabela’s CLUB Visa credit card, which serves as its primary customer loyalty rewards program. Cabela’s stock trades on the New York Stock Exchange under the symbol“CAB.”

About Activision Publishing

Headquartered in Santa Monica, California, Activision Publishing, Inc. is a leading worldwide developer, publisher and distributor of interactive entertainment and leisure products.

Activision Publishing maintains operations in the U.S., Canada, the United Kingdom, France, Germany, Ireland, Italy, Sweden, Spain, Norway, Denmark, the Netherlands, Australia, Russia, Japan, South Korea, China and the region of Taiwan. More information about Activision and its products can be found on the company’s website,

Cautionary Note Regarding Forward-looking Statements: Information in this press release that involves Activision Publishing’s expectations, plans, intentions or strategies regarding the future are forward-looking statements that are not facts and involve a number of risks and uncertainties. Activision Publishing generally uses words such as“outlook,”“will,”“could,”“would,”“might,”“remains,”“to be,”“plans,”“believes,”“may,”“expects,”“intends,”“anticipates,”“estimate,” future,”“plan,”“positioned,”“potential,”“project,”“remain,”“scheduled,”“set to,”“subject to,”“upcoming” and similar expressions to identify forward-looking statements. Factors that could cause Activision Publishing’s actual future results to differ materially from those expressed in the forward-looking statements set forth in this release include, but are not limited to, sales levels of Activision Publishing’s titles, shifts in consumer spending trends, the impact of the current macroeconomic environment, the seasonal and cyclical nature of the interactive game market, Activision Publishing’s ability to predict consumer preferences among competing hardware platforms (including next-generation hardware), declines in software pricing, product returns and price protection, product delays, retail acceptance of Activision Publishing’s products, adoption rate and availability of new hardware and related software, industry competition, rapid changes in technology and industry standards, protection of proprietary rights, litigation against Activision Publishing, maintenance of relationships with key personnel, customers, vendors, licensees, licensors and third-party developers, counterparty risks relating to customers, licensees, licensors and manufacturers, domestic and international economic, financial and political conditions and policies, foreign exchange rates, integration of recent acquisitions and the identification of suitable future acquisition opportunities, Activision Blizzard’s success in completing the integration of the operations of Activision Publishing and Vivendi Games in a timely manner, or at all, and the combined company’s ability to realize the anticipated benefits and synergies of the transaction to the extent, or in the timeframe, anticipated, and the other factors identified in the risk factors section of Activision Blizzard’s most recent annual report on Form 10-K and any subsequent quarterly reports on Form 10-Q. The forward-looking statements in this release are based upon information available to Activision Publishing and Activision Blizzard as of the date of this release, and neither Activision Publishing nor Activision Blizzard assumes any obligation to update any such forward-looking statements. Forward-looking statements believed to be true when made may ultimately prove to be incorrect. These statements are not guarantees of the future performance of Activision Publishing or Activision Blizzard and are subject to risks, uncertainties and other factors, some of which are beyond its control and may cause actual results to differ materially from current expectations.

©2009 Activision Publishing, Inc. Activision is a registered trademark of Activision Publishing, Inc.“PlayStation”,“PLAYSTATION” and“PS” Family logo are registered trademarks of Sony Computer Entertainment Inc. 2008 Microsoft Corporation. Microsoft, Xbox, Xbox 360, Xbox LIVE and the Xbox logos are trademarks of the Microsoft group of companies and are used under license from Microsoft. Wii and Nintendo DS are trademarks of Nintendo.© 2006 Nintendo. All other trademarks and trade names are the properties of their respective owners. All other trademarks and trade names are the properties of their respective owners. All rights reserved.

# # #

Shaun Norton

Sandbox Strategies

Games PressGames Press is the leading online resource for games journalists. Used daily by magazines, newspapers, TV, radio, online media and retailers worldwide, it offers a vast, constantly updated archive of press releases and assets, and is the simplest and most cost-effective way for PR professionals to reach the widest possible audience. Registration for the site and the Games Press email digest is available, to the trade only, at


text:
GFI announces the release of Precursors and Xenus II White Gold

Moscow, Russia, December, 2010– Game Factory Interactive (GFI), one of the leading Russian publishers and developers, presents Christmas and New Year gifts to all players launching the long-awaited FPS/RPG games  Precursors and Xenus II White Gold.  Have cool, exciting and unforgettable holidays with Caribbean drug mafiosos under high palms or among alien civilizations of far galaxies!!!

These games will be available on partners’ sites: Gamersgate and GameStreamer.

Precursors, predecessors, the legend of galaxy… Who are carrying the forebear’s genes among existing races, we can only suppose…

The worlds of galaxies are everlasting, beautiful, unique and dangerous. Some of them are inhabited, some are ruined and deserted, others are just smugglers’ trans-shipment stations. Only when you have passed through all these worlds, sensed the heat of blazing lava-seas and the chilling cold of abysses, seen the stark heavens, you will realize the real power of nature.

Only when you have met corrupt judges, mendacious politicians, unselfish protectors and cruel tyrants you will understand the passions of society. When you have reached unexplored sectors of the galaxy and come across mysterious natural phenomenon, have flown through the tail of a comet or dived into the casual portal, only then you will perceive the infinite depth and diversity of the universe.

Xenus II White Gold

The Caribbean islands are lands full of secrets, mystery and the spirit of romance. These islands are beautiful and… lethally dangerous. Centuries ago the Caibbean waters were teeming with pirates and adventurers... since not much has changed.

Immerse in the world of drug-dealers, bandits and Mafioso, take part in shoot-outs  and bandit fights and then reward yourself with an exotic rum or whiskey.

About Russobit-M/GFI

Russobit-M/GFI - is Russian computer games' developer, publisher and distributor - one of the acknowledged leaders in the industry. The company was founded in 1999. During the 11 years of its existence, more than one hundred projects was published under its brand; more than 150 of them were developed in Russia.

About GamersGate

GamersGate is a leading digital distribution platform creating easily accessible gaming experiences for gamers worldwide– anytime, anywhere. Viewed as a strong contender in the digital download arena, GamersGate continues to redefine the download industry and future strategies including the launch of several accompanying services and comprehensive tools, in addition to an aggressively growing product portfolio with over 2,000 games from a wide range of internationally recognized publishers.  For more information, check out the GamersGate website at

Media Content

Joe Ziemer, TriplePoint for GamersGate

(212) 730-9744

About GameStreamer, Inc.

GameStreamer’s mission is to provide a platform for the digital distribution, commerce and social engagement of games. An innovator, GameStreamer operates a B2B network with clients across the globe.

GameStreamer works with companies as White Label Partners to create custom digital game stores targeting the partners’ demographics and reflecting their brands.

Games PressGames Press is the leading online resource for games journalists. Used daily by magazines, newspapers, TV, radio, online media and retailers worldwide, it offers a vast, constantly updated archive of press releases and assets, and is the simplest and most cost-effective way for PR professionals to reach the widest possible audience. Registration for the site and the Games Press email digest is available, to the trade only, at


text:
Prototype 2 Facebook App - //Blacknet

Radical Entertainment is extremely proud to announce the launch of //Blacknet, a free and exciting new fan-focused Facebook application located at

Modeled after PROTOTYPE® 2 ’s //Blacknet in-game interface, Facebook fans will be able to work together as a community to“hack” into Blackwatch’s encrypted network and uncover new exclusive content, including behind-the-scenes file access, gameplay videos, screenshots, wallpapers, interviews, commentaries and a whole lot more!

With awesome new content unlocking every week, Radical intends to continue giving back to the PROTOTYPE fan community all the way up to and beyond PROTOTYPE 2 ’s launch in 2012.

Games PressGames Press is the leading online resource for games journalists. Used daily by magazines, newspapers, TV, radio, online media and retailers worldwide, it offers a vast, constantly updated archive of press releases and assets, and is the simplest and most cost-effective way for PR professionals to reach the widest possible audience. Registration for the site and the Games Press email digest is available, to the trade only, at


text:
Alumna profile EXTRA
About Women in Science

on this article

Fall-Winter 2012 issue (pdf)

Lucille B. Rosenberg, MD, Fel ’69Lucille B. Rosenberg, MD, Fel ’69

“You don’t retire from something. You retire to something,” says Lucille B. Rosenberg, MD, Fel ’69, whose post-practice activities illustrate the same depth of dedication to community that she demonstrated during her career. A child psychiatrist who through the years served many of the most vulnerable populations in southern Wisconsin, Dr. Rosenberg has maintained an ambitious schedule, working with youth-oriented agencies in Milwaukee.

Her input also has been particularly valuable to the Medical College of Wisconsin in its development of Women in Science, an annual lecture series and support organization that benefits from her lifetime of experience as an advocate for women in health care.

Since 2007, Women in Science has organized luncheon presentations featuring women faculty members from the Medical College who discuss their work and career paths. Membership is open to the public, and each lecture is an opportunity for the community to meet outstanding female scientists and physicians and learn about their research and its impact on health. Women in Science also provides financial support for women scholars at the Medical College to advance their research.

 “My philosophy is that we need to mentor and support women,” Dr. Rosenberg said. “I think Women in Science has done that and draws attention to women’s many roles. Women have a unique perspective in medicine.”

A member of the Women in Science Advisory Committee since its inception, she has sought to emphasize the involvement of community members, particularly practicing physicians, as well as showcase the contributions women are making to research and medicine. Women in Science also models the value of mentoring women medical students so they reach their aspirations. History informs Dr. Rosenberg’s opinion that these are important efforts, and her career journey includes moments dedicated to making a difference.

Dr. Rosenberg began medical school at University of Wisconsin-Madison in 1946, a year that saw a noticeably high number of women matriculants nationwide. Because of this critical mass, Dr. Rosenberg and her peers were able to reconstitute a chapter of Alpha Epsilon Iota, the national women’s medical sorority. It was one of her first women’s advocacy efforts, but not her last.

Dr. Rosenberg was already a mother when she finished her pediatrics residency and eventually had five children, so she knows well the juggling of responsibilities required by professional women.  She became the first woman President of the Milwaukee County Medical Society, and then advocated at the state level for a session just for women physicians, which drew more than 200 members to its first meeting.

The effort led to the creation of Women in Medicine, a collaborative organization for women physicians in Milwaukee and Madison that hosted seminars on subjects that resonated with women professionals, like work-life balance, and helped raise awareness of women’s accomplishments in medicine. The organization served as a template for the Medical College’s Women in Science program.

“We started Women in Medicine to help communication among women in practice and with women medical students so they felt like they had mentors,” she said.

Dr. Rosenberg is a former member of the pediatrics and psychiatry faculty at the Medical College of Wisconsin. She practiced at Children’s Hospital of Wisconsin in a psychiatric outpatient setting and also served as Medical Director at Curative Care Network, working primarily with children with multiple disabilities.

In the 1980s, Dr. Rosenberg joined Sinai Samaritan as Medical Director of the Child and Adolescent Outpatient Clinic. She retired in 1997. Throughout her career, she found the opportunity to work in a multidisciplinary setting to be among the most gratifying aspects.

“We can always learn something from one other,” she said. “I’ve certainly learned much from my colleagues.”

About Women in Science

Women researchers and physicians at The Medical College of Wisconsin are making discoveries that are saving lives and improving treatments for patients with injuries and complex diseases. The mission of Women in Science is to showcase outstanding research and provide financial support for women scientists at the Medical College of Wisconsin.

Learn more about the Medical College of Wisconsin’s Women in Science program by accessing these resources:

Comments are subject to approval. The Medical College reserves the right to edit comments for length, grammar, clarity and appropriateness.

Please include your first and last name. Alumni, please also feel free to include your class year(s). Your e-mail address will not be published.

Comments / Ratings

Thank you
Lynn Ryan
12-12-12  5:36:37 AM

Thank you, Lucy, for your continuing interest in helping the needy you meet. We've come a long way since my interview at Marquette Med School in 1963 when I was told they only had openings for female Maryknoll missionaries! I thank God for your good health. Lynn Ryan, MD. UW 1968

The following required items were not provided or are in the wrong format. Please provide the required responses and submit again:

  Please enter your name
  Please enter a valid email
Comment Title:
Comment: 550 characters left
  Please enter a comment

Medical College of Wisconsin
8701 Watertown Plank Road
Milwaukee, WI 53226
(414) 955-8296
Directions & Maps
© 2015

Page Updated 11/27/2012


text:
Cancer Center Scientific Retreat

Three $500 Travel Grants Awarded to Best Oral Presentations

The first annual Cancer Center Scientific Retreat is on Friday, May 3rd, from 8:00 AM to 4:30 PM at American Serb Hall, 5101 W. Oklahoma Avenue, Milwaukee, WI.  Breakfast and a hot buffet lunch will be provided, so all attendees must register for this event.

The focus of this year’s retreat is cancer disparities research and community outreach.  Edward E. Partridge, MD, is the key-note speaker. Dr. Partridge is Director of the University of Alabama at Birmingham Comprehensive Cancer Center.  He is a nationally recognized leader in cancer control and disparities research, and currently serves on our External Scientific Advisory Board (ESAB). Learn more about Dr. Partridge, or follow his blog.

Other featured speakers on health disparities and community outreach include Dr. Syed Ahmed, Senior Associate Dean for Community Engagement, Dr. Alanzo Walker, Senior Associate Dean of Faculty Affairs and Diversity, and Dr. Reza Shaker, Senior Associate Dean for Clinical and Translational Research and Director of the CTSI

The retreat will also include research program speaker and poster sessions selected from submitted abstracts. All of the oral presentations given at the Scientific Retreat will be evaluated by a team of judges.  The three winning presentations will be announced at the end of the retreat, and those presenters will each be awarded a $500 travel grant from the MCW Cancer Center.

If you have any questions, please contact Michele Ward at or (414) 805-8278.

We look forward to this event and opportunities for collaboration.

Medical College of Wisconsin
8701 Watertown Plank Road
Milwaukee, WI 53226
(414) 955-8296
Directions & Maps
© 2015

Page Updated 04/02/2015


text:
Quest Magazine

ALS Stem Cell Trial is Moving Forward

The Maryland biotherapeutics company Neuralstem will move its clinical trial of neural stem cells in people with amyotrophic lateral sclerosis (ALS) into its next stage, following a thumbs-up from the trial's safety monitoring board, the company announced Oct. 18, 2010. (See Neuralstem Updates ALS Clinial Trial Progress.)

Two-Drug Combo Makes a Difference in FA

Results from a clinical trial conducted in Spain involving 20 individuals with Friedreich's ataxia (FA) show that a combination therapy using two drugs, idebenone and deferiprone (DFP), demonstrated a "stabilizing effect" in neurological function and reduced abnormal heart-muscle enlargement in participants who received the two-drug combo for a period of 11 months.

Research Briefs: DMD

Anti-myostatin drug trial shows good preliminary results

National ALS Registry Now Open

After two years of laying the groundwork, the Agency for Toxic Substances and Disease Registry (ATSDR) has opened its National ALS Registry to collect information from people living with ALS.

Scientists at the ATSDR, which is part of the Centers for Disease Control and Prevention in Atlanta, hope registry data will provide insight into the causes of, and potential treatments for, ALS.

Misfolded SOD1 Found in Sporadic ALS Samples

A new study suggests misfolding of the SOD1 protein may be a common contributor not only to familial (inherited) amyotrophic lateral sclerosis (ALS) caused by mutations in the SOD1 gene but to the more common sporadic (noninherited) form of the disease as well.

Intravenous AVI4658 Shows Safety, Benefit in DMD

The experimental drug AVI4658, in development by AVI BioPharma to treat Duchenne muscular dystrophy (DMD) caused by specific genetic mutations, was well tolerated and resulted in increased production of the needed dystrophin protein. Measured aspects of cardiac, pulmonary and skeletal muscle function remained stable.

Low-Dose Ataluren Shows Some Benefit in DMD/BMD

A low-dose regimen of ataluren (formerly called PTC124), an experimental drug developed by PTC Therapeutics to treat  Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) caused by a certain type of genetic mutation, is superior to a high-dose regimen or a placebo.

ALS TDI Conference Summarizes ALS Research Progress

Themes discussed throughout the research symposium included ALS genetics, clinical trials, the role of the immune system in disease onset and progression, biomarkers, and the use of stem cells in ALS therapeutics.

Diaphragm Pacing System Awaiting FDA Approval for ALS

The Synapse Biomedical NeuRx Diaphragm Pacing System (DPS), a device that stimulates the respiratory diaphragm with electrical signals, has received Humanitarian Use Device designation from the U.S. Food and Drug Administration (FDA), allowing it to be used by people with amyotrophic lateral sclerosis (ALS) who have a “stimulatable” diaphragm and are experiencing chronic breathing problems.

Comic with BMD Builds Accessibility One Doorway at a Time

Jonah Bascle (rhymes with “rascal”) unleashes his zany brand of stand-up comedy on New Orleans nightclub and restaurant patrons four or five times a week. As an unusual consequence of these gigs, more and more of the historical city’s old buildings are becoming accessible to people in wheelchairs.

As a guy who uses a wheelchair himself due to Becker muscular dystrophy, Bascle, 24, knows first hand how difficult it is to navigate the entrances to many long-established buildings in the legendary home of Mardi Gras.


text:
ijms International Journal of Molecular Sciences Int. J. Mol. Sci. 1422-0067 Molecular Diversity Preservation International (MDPI) 10.3390/ijms11051991 ijms-11-01991 Communication Synthesis of 4′,7-Diacetoxyapigenin and Its Apoptotic Induction in Human Hep G2 Cells XuKeyong1* LiuFeng2 LiuBenguo3 GaoHan3 NingZhengxiang1 College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640, China; E-Mail: fezhning@scut.edu.cn (Z.N.) The Key Laboratory of Chemical Biology, Guangdong Province, Graduate School at Shenzhen, Tsinghua University, Guangdong 518055, China; E-Mail: liu.feng@sz.tsinghua.edu.cn (F.L.) School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China; E-Mails: zzgclbg@126.com (B.L.); gh@hist.edu.cn (H.G.) Author to whom correspondence should be addressed; E-Mail: kyxky@163.com; Tel.: +86-20-87111910; Fax: +86-20-87113848. 30 4 2010 2010 11 5 1991 1998 30 3 2010 23 4 2010 © 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. 2010

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

In this study, 4′,7-diacetoxyapigenin [4-(7-acetoxy-5-hydroxy-4-oxo-4H-chromen-2-yl) phenyl acetate] was synthesized for the first time. Its chemical structure was identified by UV, ESI-MS, 1H and 13C-NMR. It could inhibit the proliferation of Hep G2 cells in a dose-dependent manner and induce the significant increase of the G0/G1 cell population. After treatment by 4′,7-diacetoxyapigenin, phosphatidylserine of Hep G2 cells could significantly translocate to the surface of the membrane. The increase of an early apoptotic population was observed by both annexin-FITC and PI staining. It was concluded that 4′,7-diacetoxyapigenin not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle.

apoptosis cell cycle Hep G2 4′,7-diacetoxyapigenin

Apoptosis is a highly regulated cell death process with characteristic biochemical features [1,2] and membrane-bond apoptotic bodies [3]. It occurs both during normal development and under certain pathological conditions in metazoans, and plays a crucial role in the maintenance of tissue homeostasis by the selective elimination of excessive cells [4,5]. Evasion of apoptosis is an essential hallmark of cancer [6]. Genetic changes resulting in loss of apoptosis or derangement of apoptosis-signalling pathways in the transformed cells are likely to be critical components of carcinogenesis [7,8]. Killing tumour cells through the induction of apoptosis has been recognized as a novel strategy for the identification of antitumour drugs and a valuable tool for cancer treatment [9,10]. In this study, in order to determine the effect of the phenolic hydroxyl groups at C-4′ and C-7 of apigenin on its anticancer activity, 4′,7-diacetoxy apigenin [4-(7-acetoxy-5-hydroxy-4-oxo-4H-chromen-2-yl) phenyl acetate] was synthesized for the first time and its apoptotic induction in human Hep G2 cells was evaluated.

Results and Discussion Identification of 4′,7-Diacetoxyapigenin

Using UV, ESI-MS and NMR, the flavonoid obtained in this study were identified as 4′,7-diacetoxyapigenin (Figure 1).

Anti-Proliferation Activity of 4′,7-Diacetoxyapigenin on Hep G2 Cells

The inhibitory effects of 4′,7-diacetoxyapigenin on the proliferation of Hep G2 cells were tested at different concentration for 48 h and the inhibition rate (IR) was determined (Figure 2). It induced a dose-dependent inhibitory effect. The inhibitory concentration 50% (IC50) was about 73.7 μM. The effect of 4′,7-diacetoxy apigenin was far low than that of apigenin (about 29.7 μM) [11]. Apigenin was more potent in the inhibition of Hep G2 cells proliferation than that of 4′,7-diacetoxyapigenin, which could mean the presence of the phenolic hydroxyl groups at C-4′ and C-7 of apigenin was very important for its anticancer activiy. The effect of 4′,7-diacetoxyapigenin on the cell morphology is shown in Figure 3. It could be found that the viable cells could remain their common shape, but non-viable cells had lost their shape. And necrosis could also contribute to the induced cell death.

Flow Cytometry Analysis of Cell Cycle and Cell Apoptosis

To determine whether the Hep G2 cells treated with 4′,7-diacetoxyapigenin undergo apoptosis, the cell distribution in the cell cycle was examined by PI staining. The significant increase of the population in the G0/G1 phase in Hep G2 cells could be observed and the population of cells in other phases of the cell cycle remained unaffected.

The hallmark of early apoptotic cells is the transverse redistribution of plasma membrane phosphatidylserine (PS) [12]; thus, the annexin V binding assay was performed to detect the surface exposure of PS. In this study, the Hep G2 cells were treated with 4′,7-diacetoxyapigenin (70 μM) for 48 h. Figure 5 shows the FACS histogram with dual parameters including annexin V-FITC and PI. The dual parametric dot plots combining annexin V-FITC and PI fluorescence show the viable cell population in the lower left quadrant (annexin V-negative/PI-negative), the early apoptotic cells in the lower right quadrant (annexin V-positive/PI-negative), and the late apoptotic cells in the upper right quadrant (annexin V-positive/PI-positive). In untreated Hep G2 cells, 0.7% of cells were annexin V-positive/PI-negative, 0.1% of cells were both annexin V- and PI-positive. In treated Hep G2 cells, the annexin V-positive/PI-negative and double-positive cells increased to 54.1% and 32.9%, respectively. The result suggested that 4′,7-diacetoxyapigenin could induce cells into an apoptotic pathway resulting in the inhibition of proliferation of Hep G2.

Experimental Section Materials and Instrumentation

Apigenin (Purity 95%) came from our previous study [13]. A human hepatoma cell line (Hep G2) was obtained from the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. Powdered Dulbecco modified eagle medium was purchased from GIBCO (Grand Island, NY, USA). Foetal bovine serum (FBS) and antibiotics (penicillin and streptomycin mixture) were purchased from Hyclone Laboratories, Inc. Propidium iodide (PI) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. An annexin V-FITC apoptosis detection kit was purchased from CLONTECH Company. UV analysis was performed on a TU-1810PC UV-visible spectrophotometer (Purkinje, China). 1H- and 13C-NMR spectra were recorded in DMSO-d6 using an AVANCE Digital 400 MHz NMR spectrometer (Bruker, Germany). ESI-MS analysis was taken on a ZQ 2000 electrospray ionization mass spectrometer (Waters, USA) in the positive ion mode.

Synthesis of 4′,7-Diacetoxyapigenin

Acetic anhydride (0.94 mL, 10.0 mmol) was added dropwise to a solution of apigenin (1.35 g, 5.00 mmol) in dry pyridine (80 mL) at room temperature. After stirring the mixture for 24 h at room temperature, it is poured into ice-cold water (1,600 mL). The white precipitate is separated by filtration, washed twice with a small amount of ice-cold water and recrystallized from methanol (300 mL). The resultant material is washed with distilled water and dried at 60 °C to yield the product (0.842 g) as a slightly white solid. UV, λmax(nm) (MeOH) 270, 300sh; ESI-MS2 negative ion m/z: 747 ([2M+K]+), 731 ([2M+Na]+), 709 ([2M+H]+), 393 ([M+K]+), 377 ([M+Na]+), 355 ([M+H]+), 313 ([M-acetyl+H]+), 271 ([M-2acetyl+H]+); 13C-NMR δ, 183.02 (C-4), 169.35 (C-2″/2″′), 168.89 (C-2), 163.96 (C-5), 161.20 (C-7), 156.72 (C-9), 153.98 (C-4′), 128.65 (C-1′), 128.37 (C-2′/6′), 123.17 (C-3′/5′), 108.64 (C-6), 106.17 (C-10), 105.87(C-3),102.14 (C-8), 21.37 (C-3″/3″′); 1H-NMR δ, 12.87 (s, 5-OH), 8.16 (d, J = 8.79 HZ, H-2′/6′), 7.37 (d, J = 8.79 HZ, H-3′/5′), 7.14 (s, H-8), 7.10 (d, J = 1.99 HZ, H-3), 6.67 (d, J = 1.99 HZ, H-6), 2.32 (s, H-3″/3″′).

Cell Culture and Drug Treatment

Hep G2 cells were cultured in DMEM medium with 10% FBS, 100 UI/mL penicillin and 100 μg/mL streptomycin in humidified air at 37 °C with 5% CO2. Exponentially growing Hep G2 cells were collected and re-suspended in fresh medium for 4 h and then exposed to various concentrations of 4′,7-diacetoxy apigenin.

MTT Assay

Survival of cells was evaluated by using a system based on MTT, which was reduced by living cells to yield a soluble formazan product that could be detected colorimetrically. Cells were suspended in 96-well plates of 90 μL medium at a density of 2 × 104 cells/well and 10 μL 4′,7-diacetoxyapigenin in different concentrations. These were then incubated in humidified air at 37 °C with 5% CO2 for 48 h, exposed to 10 μL MTT (5 mg/mL) and incubated for another 4 h under the conditions mentioned above. The formazan precipitate was dissolved in 100 μL DMSO. IC50 values were tested through the MTT method [14]. The inhibition rate (IR%) was calculated as follows: IR% = (mean control absorbance-mean experimental absorbance)/mean control absorbance ×100%.

Flow Cytometry Analysis

The flow cyctometry analysis was performed on a FACS Calibur Flow cytometer (BeckmanCoulter, USA). Cell pellets were fixed in 70% ethanol at −20 °C for at least 12 h or overnight. After being washed twice with ice-cold PBS, they were incubated in RNase A/PBS (1 mg/mL) at 37 °C for 30 min, and stained with PI (0.5 mg/mL) at room temperature for 15 min. The intracellular DNA was then labelled with PI and the PI fluorescence of individual nuclei determined by a FACSCalibur fluorescence-activated cell sorter at 488 nm excitation. Surface exposure of phosphatidylserine in apoptotic cells was measured by the annexin V-FITC apoptosis detection kit according to the manufacturer’s instructions. Additional exposure to PI made it possible to differentiate the early apoptotic cells (annexin V-positive/PI-negative) from the late apoptotic cells (annexin V-positive and PI-positive).

In this study, 4′,7-diacetoxyapigenin [4-(7-acetoxy-5-hydroxy-4-oxo-4H-chromen-2-yl) phenyl acetate] was synthesized for the first time. It could inhibit the proliferation of Hep G2 cells in a dose-dependent manner. By using flow cytometry, it was found that 4′,7-diacetoxyapigenin not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle.

The financial support provided by the Foundation of Henan Educational Committee (No. 2009A550005) and the Science and Technology Plan of Henan institute of science and technology (No. 7040) was greatly appreciated.

References and Notes JacobsonMDWeilMRaffMCProgrammed cell death in animal developmentCell19978834735410.1016/S0092-8674(00)81873-59039261 NagataSApoptosis by death factorCell19978835536510.1016/S0092-8674(00)81874-79039262 KiddVJProteolytic activities that mediate apoptosisAnnu. Rev. Physiol19986053357310.1146/annurev.physiol.60.1.5339558476 ThompsonCBApoptosis in the pathogenesis and treatment of diseaseScience19952671456146210.1126/science.78784647878464 NayfieldSGKarpJEFordLGDorrFAKramerBSPotential role of tamoxifen in prevention of breast cancerJ. Natl. Cancer Inst1991831450145910.1093/jnci/83.20.14501920492 HanahanDWeinbergRAThe hallmarks of cancerCell2000100577010.1016/S0092-8674(00)81683-910647931 KastanMBCanmanCELeonardCJCell cycle control and apoptosis: implications for cancerCancer Metastasis Rev19951431510.1007/BF006902077606818 Schulte-HermannRBurschWLow-BaselliAWagnerAGrasl-KrauppBApoptosis in the liver and its role in hepatocarcinogenesisCell Biol. Toxicol19971333934810.1023/A:10074956268649298254 SmetsAProgrammed cell death (apoptosis) and response to anti-cancer drugsAnticancer Drugs19945398186426 KornblauSMThe role of apoptosis in the pathogenesis, prognosis, and therapy of hematologic malignanciesLeukemia199812S41S469777895 ChiangLNgLTLinIKuoPLinCAnti-Proliferative effect of apigenin and its apoptotic induction in human Hep G2 cellsCancer Lett200623720721410.1016/j.canlet.2005.06.00216023288 Van EngelandMNielandLJWRamaekersFCSSchutteBReutelingspergerCPMAnnexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposureCytometry1998311910.1002/(SICI)1097-0320(19980101)31:1<1::AID-CYTO1>3.0.CO;2-R9450519 LiuBNingZGaoJXuKPreparing Apigenin from Leaves of Adinandra nitidaFood Technol. Biotech200846111115 MosmannTRapid colorimetric assay for cellular growth and survival: Application to proliferation and cytoxicity assaysJ. Immunol. Methods198365556310.1016/0022-1759(83)90303-46606682 Figures

Chemical structure of 4′,7-diacetoxyapigenin.

Effect of 4′,7-diacetoxyapigenin on Hep G2 cell proliferation.

Morphology of Hep G2 cells treated by 4′,7-diacetoxyapigenin (70 μM, 48 h).

Effect of 4′,7-diacetoxyapigenin on cell cycle distribution in Hep G2 cells.

Flow cytometric analysis of phosphatidylserine externalization (annexin V binding) and cell membrane integrity (PI staining) in Hep G2 cells undergoing apoptosis.