The Nigeria Immigration Service (NIS) has denied playing any part in the process that led to a known ISIS leader in Lebanon receiving a visa to visit Nigeria. Rather, it was the Minister of Foreign Affairs, which is under the supervision of President Muhammadu Buhari, that issued the visa.
In August, The Trent was the first newspaper in Nigeria to report that a radical Muslim cleric, Ahmad al-Assir had been arrested by Lebanese authorities as he attempted to leave Lebanon to Nigeria via Cairo.
Assir was apprehended while attempting to travel with a fake Palestinian passport and a valid Nigerian visa at the Beirut’s Rafik Hariri International Airport early Saturday, August 15, 2015 in Lebanon. He was travelling with another man who was also with a Nigerian visa.
When The Trent reported the arrest of Al Assir with a Nigerian visa in Lebanon, President Buhari ordered a probe on how Assir obtained a Nigerian visa. “The reported arrest of the wanted terrorist is a huge embarrassment to Nigeria and the President has directed that the matter should be investigated,” the ministry official told Vanguard on condition of anonymity.
Foreign Affairs Ministry Bypasses NIS
The Comptoller-General of the NIS, Martin Abeshi revealed this new and important piece of information to news media in a press conference in Abuja on Wednesday, September 30, 2015. He also revealed that two men connected to Al Assir, whom the fugitive ISIS cleric was visiting in Nigeria have been tracked down and arrested in Kano.
The men, who have not been identified, have been handed over to the National Security Adviser, General Babagana Monguno (Rtd).
“From the record available, the Immigration Service did not issue that visa. It might be the officials of the foreign affairs ministry because they also issue visa on our behalf,” Abeshi revealed.
The Immigration boss also said that the NIS swept into action when the news of the arrest of the ISIS cleric in Lebanon broke and arrested the individuals connected with the fugitive, Assir’s visit to Nigeria.
“When we were told that the person [Al Assir] was heading to Nigeria, the people the man was coming to visit were identified by our men. Our officers in Kano went into action and arrested the two of them immediately and handed them over to the National Security Adviser,” he said.
The status of the probe ordered by President Buhari into this threat to Nigeria’s national security has not been made public.
Two Years A Fugitive
New face of IS linked fugitive cleric, Ahmad al-Assir who was en route Nigeria via Cairo. pic.twitter.com/eT45QGg5es
— Mr. Aye Dee (@MrAyeDee) August 16, 2015
Prior to his arrest, Sheikh Assir has been on the run for two years. He was declared wanted in Lebanon in 2013 when his followers clashed with the Lebanese army killing at least 18 soldiers.
The radical cleric was relatively unknown prior to the outbreak of the Syrian war in 2011. But, he quickly gained notoriety as a “a self-proclaimed defender of Sunni rights” due to his fiery anti-Hezbollah rhetoric. Hezbollah is Lebanon’s Shia movement which backs Syria’s President Assad. He had been recruiting his followers to join the Islamic State terrorist group (ISIS) in Syria fighting against Assad’s government.
Violence broke out in 2013 when one of Assir’s men was caught with unlicensed weapons in his car at a military checkpoint in Sidon, south of Beirut. In reaction, Assir’s followers opened fire on the soldiers manning the checkpoint sparking a two-day battle between his militants and the army.
According to reports, machine guns and rockets were used on the Lebanese troops by Assir’s men in fierce fighting which ended with at least 18 soldiers and 40 militants dead. Heavy weapons and military-style uniforms were discovered in Assir’s compound.
Assir is regarded as media savvy and is known for his publicity stunts.
Implications For Nigeria’s President Buhari
Alexander Mukhtar Dan’Iyan, the editor-in-chief of the 15Past8 Media Group based in New York City, who tweets via @MrAyeDee on Twitter, says that Assir is the ”unofficial de-facto Emir of ISIS in Lebanon”.
Ahmed al-Assir is the unofficial defacto IS Emir for Lebanon. Only reason he's unofficial is because Bay'ah has not been publicly accepted.
— Mr. Aye Dee (@MrAyeDee) August 16, 2015
The Trent raised serious questions for the Nigerian government because of the twin issues of Assir’s fugitive status and his links with ISIS.
How would a radical Muslim cleric, a fugitive linked to ISIS be granted a Nigerian visa on a fake Palestinian passport? What was Assir’s mission in Nigeria? Who are his contacts in Nigeria? Who was he communicating with in Nigeria? Where was he planning to stay in Nigeria? Most importantly, who granted his visa and based on what recommendations?
The incursion of ISIS into a Boko Haram ravaged Nigeria gives Nigerian authorities more cause to worry as Boko Haram attacks continue to increase since President Muhammadu Buhari assumed office on May 29, 2015.
In March 2015, Richard Grenell, a former spokesman for four United States Ambassadors to the United Nations, raised an alarm about the dangers of a Buhari presidency for Nigeria and the whole of Africa.
In an article titled “Nigeria on the brink” which was published in the Washington Times, a US based publication, Grenell observed that the former military Head of State was working tirelessly to introduce the Sharia law across the country, which according to him would further pose a major challenge to the fight against terrorism in Nigeria and Africa in its entirety.
ALSO READ: Nigeria on the brink – Washington Times
According to Grenell, “Western foreign policy observers pre-occupied with the rise of ISIS in the Middle East should wake up to the reality unfolding in Nigeria. Opposition candidate General Buhari wants Sharia law throughout Nigeria. In fact, he wants it everywhere.
“Is this an indication that Buhari supports violence because the end goal of the terrorist attacks throughout Nigeria and Africa is an Islamic state? It is a legitimate question that should be asked by leaders of the Obama administration.
“Boko Haram has pledged its allegiance and support to ISIS. The Northern Nigerian based Islamic terrorist group wants Sharia law throughout the country and beyond. They are also actively terrorising Chad, Niger and Cameroon with their goal of an Islamic state.
“Buhari has also spoken sympathetically about members of the terrorist group Boko Haram, has cautioned against a rush to judgement on its members and has personally been selected by the terrorist group to lead its negotiations with the Government of Nigeria.
“Buhari’s election as Nigeria’s head of state would be a disaster for Africa. It would also signal trouble for the West’s fight against ISIS and terrorism throughout the Middle East.”
Additional reports by Yemi Adebowale.


text:
The Wizard's Tower - Vue
Created by: Dark Anvil
File Size: 17.7mb
Constructed from a sketch concept by fantasy designer Joe Bailey.
The Wizards Tower can add that dramatic extra to your fantasy renders.
It can carry a whole scene as the only building, or blend into
the background as your artistic desire demands.
We have resisted the temptation to post a picture of the Wizard battling fiercely with a distinctly grumpy dragon. We want you to do that.
This is the Vue Version
Get the Sacrificial Altar, Blacklight Forge, Chalice Tower, Kroluk Distillery, Kroluk & and Stables, Troll House and Wizards Tower for $5 and save $8.93. A similar deal is avaliable in Poser Format.
Click here for the bundles page.
Additional Product Images:
|Dark Anvil licence. A copy can be found here.
Daz Studio Versions:
I'm pleased to announce that the nice people at Vanishing Point are now converting these models
to native studio format.
Please note as I don't host copies of these 'new' studio versions, these ones are not included
in any bundles, deals or repeat downloads avaliable here. These are only sold via VP, or by VP at
selected brokeages and not here. Prices may be different between stores as well, this is because each store operates
it's own pricing stratgies and practices.

domain: artistic


Everyday in the morning, the company is losing Internet connectivity. In order to restore it, sometimes i can simply restart the firewall service, sometimes I need to disable/enable the internal NIC, and sometimes i need to reboot. When this happens I cannot ping the internal IP but I can RDP to the box, which is strange. This all seems to be related to the number of connections to the DC but i cannot be sure. Things I've tried:
- Rebuild the cache file
- Disable the Flood Mitigation Settings
- Increase the timeout for Connectivity Verification (see Error below)
None of this seems to do anything. Here are some log and event viewer entries i'm seeing:
ISA Server Detects routes through the network adapter that do not correlate with the network to which this network adapter belongs...
The routing table for the network adapter Internal includes IP address ranges that are not defined in the array-level network.
Windows cannot determine the computer name. Group policy aborted. The user in this case was the ISA admin account. After this error, the internet was lost.
The number of concurrent TCP connections from the source IP address .... exceeded the configured limit. (This seems to come up after a firewall service restart)
I get alot of Concurrent TCP connections from One IP address alerts. Systems do not seem to be compromised though as the IP address is never consistently the same. Granted they are DHCP so they may just be pulling different addresses. One of them is my machine actually. Once that I see though is from an outside IP address.
Connectivity Verifier "DNS" (and "DHCP") reported an error when trying to connect to (internal DC). The request timed out.
Could I have a DNS issue?
ISA Server detected routes through the network adapter Internal that do not correlate with the network to which this network adapter belongs.
The IP address it gives for this error are in our DHCP scope so not sure why this error happens.
All in all, once i restart the services, everything is ok for the day. Then the next day comes and boom, same thing.
Driving me crazy.


text:
Safaris are the most wonderful things to do if someone wants to do adventurous things. And especially the wildlife sanctuary is amazing in all the safaris. In our country, India there are many places where people can do adventurous safari and also make beautiful and amazing memories. Here are some of the best wildlife safaris which will be loved by the people and they are:-
Jhalana Leopard Safari Park, Jaipur, Rajasthan
The Jhalana Leopard safari park comes under the adventurous places of Jaipur. People can spot Leopards, striped Hyenas, Chitals, Desert Foxes and Golden Jackals in the safari. People can also spot different kinds of birds like Owl, Dusky Eagle, Indian Pitta and spotted owl. People can visit here for a leopard safari in Jaipur with their family and friends. Inside this park, there is a small local temple which is a tourist attraction. Here people can spot more than 30 leopards. There are so many beautiful spots for clicking amazing photographs. There are also so many beautiful scenic views and people will fall in love by seeing it. This safari would be considered as the best suited place in Rajasthan for nature lovers. This safari is also best for photography of the animals and birds here.
Ranthambore National Park, Rajasthan
The Ranthambore National Park is a wonderful national park spread over 1,334 square kilometers. This is one of the royal homes for the tigers. People can also see the other animals in this wonderful national park such as sloth bears, wild boars, Sambhar and many more. Suitable time to visit this wonderful National park will be from October to June.
Hemis National Park
Hemis national park is one of the largest National Parks in South Asia. This National Park is popular because it is a royal and protected home for endangered species like Tigers, Tibetan Wolf, Leopards, Eurasian Brown Bear, Red fox and Asiatic ibex. Here tourists would enjoy a peaceful and pollution free environment. Suitable time to visit this wonderful National park will be from May to September.
Jawai Leopard Reserve-Pali, Rajasthan
The majestic Jawai hills located in the Pali district of Rajasthan gives one of the best leopard safari. Leopards roam freely anywhere and this place is also known as the ‘Hills of Leopards’. This place is not only famous for leopards but also for bird watching. People can also see the striped hyenas, jackals, feral cats and wolves. There is also a temple built in a cave called Devgiri. This temple is dedicated to the Kalka Devi. Leopards are seen wandering around this temple. People will love them. sightseeing of this place and will fall in love with its majestic beauty. This safari would be considered as the best suited place in Rajasthan for nature lovers. This safari is also best for photography of the animals and birds here.
Bandhavgarh National Park, Madhya Pradesh
The Bandhavgarh National park is one of the most wonderful national parks where people can see the Bengal tigers. This national park is counted among the best national parks to do safari. Suitable time to visit this wonderful National park will be from October to June and from March to May.
Kumbhalgarh Wildlife Sanctuary- Rajsamand, Rajasthan
The Kumbhalgarh wildlife sanctuary is an amazing wildlife sanctuary situated in the Rajsamand district of Rajasthan. This sanctuary covers an area of 610.5 square km. This safari can be explored either by jeep, trekking or by riding a horse. People can visit here with their friends for trekking. Here people can spot so many animals apart from leopards and they are sambhar, nilgai, Greg jungle fowl and many more. Leopard preys on sambhar and nilgai for his food. There are also so many beautiful scenic views and people will fall in love by seeing it.
This place is adventurous as it allows people to trek here which would be dangerous for non-experienced trekkers. It is advised that only experienced trekkers can do trek here because non-experienced trekkers are not trained enough to do trek here. But basic mountaineering skills should be learned by everyone as it helps a lot in trekking, hiking,rock climbing and for many more adventurous sports. This safari would be considered as the best suited place in Rajasthan for nature lovers. This safari is also best for photography of the animals and birds here.
Sasan Gir Wildlife Sanctuary
The Sasan Gir wildlife sanctuary is a wonderful national park located on the western Gujarati peninsula. This national park will be explored in the jeep. This national park is the only national park in the world where people will find the Asiatic lions. People can also spot leopards, Jack ala, Striped Hyenas and Indian fox. This is also the home of birds and reptiles. Suitable time to visit this wonderful National park will be from December to April. This national park is closed during the monsoon season i.e. from mid- June to mid- September.
So here is all the information about the places to do safari in India. All the above places are best suited for safari and people should visit all the above places to enjoy the safari. Everyone should visit all the above places to enjoy the adventurous safari. Do not forget to taste the delicious and local food of all the above places. The above safaris would be considered as the best suited place in India for nature lovers. The above safaris are also best for photography of the animals and birds here. It is advised that those who suffer from any serious disease should not do this safari as it would be dangerous for them. People should visit all the above places once in their lifetime to make wonderful and creative memories. Click the beautiful and amazing pictures and make funny videos of the charming moments. There would be a minimal amount of entry fee to do safari in the above national parks. People will love and enjoy the safari in all the above places because of their charming beauty and dangerous animals.

domain: geography


Wrap newborns babies could make them develop anatomical problems as they remain with their legs close together for long periods. The present invention provides a solution to wrap and protect newborns and babies up to six-months, avoiding the development of anatomical problems.
This technology has been developed by a multidisciplinary team of the UTPL (Universidad Técnica Particular de Loja) where a patent application has been filed. The objective of the company represented is to find business partners interested in co-developing or licensing the patent of this invention to bring this innovation to the market.
Institution: Universidad Técnica Particular de Loja
Protection status: Patent application
Contact: Laura Núñez / firstname.lastname@example.org


text:
From The Voice of the Martyrs (VOM) newsletter, November 2013, page 3-5, by P. Todd Nettleton. See website at www.persecution.org
Most Americans have seen the graphic images of bombed-out Syrian cities and citizens killed by chemical weapons. But amid these stories of chaotic civil war and tragedy is another, less publicized story. Christian churches in Syria continue to spread hope in the face of hopelessness, holding high not a political or denominational banner but the banner of Jesus Christ– the only path to salvation and peace with God.
Before the outbreak of war, an evangelical church in one of Syria’s largest cities held several services a week for worship and prayer. Today, it holds twice as many services, and most are standing room only. A VOM contact who recently visited a Syrian church told us that his first thought upon entering was that he’d accidentally walked into a mosque. He saw so many headscarves and long beards that it appeared to be a crowd of Muslims!
VOM contacts are reporting many encouraging stories of Muslims coming to know Christ in a variety of ways. More than 70 Muslim families have turned to Christ in an area of Syria where only a dozen or so Christians existed 18 months ago. A former mullah who watched Muslim radicals from different Islamic sects kill each other while shouting “Allah is great!” began to wonder, “What kind of god are we worshiping?” Then he visited a church and learned about the God who doesn’t demand killing, but rather sacrificed his own son for our sake. His heart was moved to follow Jesus. A woman who had been paralyzed for 10 years from a stroke testified that she was healed after a Christian friend prayed for her in Jesus’ name. After experiencing his power, she gratefully committed her life to the Jesus who heals.
Syrian Christians often face persecution from their own families. A military officer who had converted to Christianity was forced into hiding when members of his family tried to have him killed for bringing shame on the family name. Despite the threats, he said he would never give up his faith. These are just a few examples of how the Holy Spirit is working among Syrians who are facing unthinkable hardships.
“When somebody comes to Christ, individually, it makes a big difference,” said one of VOM’s Syrian contacts. “When you hear about one Muslim coming to Christ, it’s a great thing, and everybody rejoices. Today in Syria I’m not talking about one person. We’re talking about hundreds, and even thousands of Muslims coming to know Christ.”
Still, our Christian brothers and sisters are no less affected by the horrors and hardships of war than other Syrians. A Syrian pastor told VOM workers that he has electricity only three hours per day and that many other parts of the country are worse. Recently, a group of Christians driving along a 37-mile stretch of road encountered 23 checkpoints, manned by armed fighters from different sides of the conflict and even different factions within each side. Each nerve-wracking checkpoint provides the potential for trouble and death, and the paperwork or dropped name that got you through the last checkpoint might raise eyebrows or gun barrels at the next.
The civil war in Syria is serving to strengthen the Church and tear down denominational barriers. Christian groups that five years ago might have questioned each other’s salvation are now gathering for weekly prayer, asking God to move in their nation and in their churches. Through prayer and fellowship, they are becoming true representatives of Christ’s Body in Syria.
VOM equips, helps and encourages these Christians who continue to minister and reach out even as their country is ripped apart by bombs and hatred. While VOM provides humanitarian assistance to Christians affected by this crisis, our principal focus will always be to empower and equip the Church to represent Christ even amid suffering. With the help of our church partners in Syria, VOM distributes New Testaments, Bibles and study Bibles this year. We are also working to provide material assistance to those who have lost much in the war.
VOM continues to support and equip evangelists who are sharing the gospel in Syria as well as those reaching out to Syrian refugees outside the country. We are working with Christians in neighboring countries to provide material assistance and gospel materials to Syrian refugees in those countries. “They are seeking hope, and they have nothing,” a VOM contact said. “They lost their house. They lost family members. They lost some of their children. And they are holding onto the hope that we have through Jesus Christ.”
Philippians 1:29-30 — For it has been granted to you on behalf of Christ not only to believe in him, but also to suffer for him, since you are going through the same struggle you saw I had, and now hear that I still have.
I Peter 4:12-13 — Dear friends, do not be surprised at the fiery ordeal that has come on you to test you, as though something strange were happening to you. But rejoice inasmuch as you participate in the sufferings of Christ, so that you may be overjoyed when his glory is revealed.
I Peter 4:16 — …If you suffer as a Christian, do not be ashamed, but praise God that you bear that name.
VOM prayer request: Please pray with us for our Christian brothers and sisters in Syria. Pray for an end to the violence and bloodshed and for God’s protection and encouragement of Christians and churches. Pray that many more Muslims will see the true face of Islam and seek the loving face of Christ.
“You are not far away, my friends. You are not. If you are a Christian, you will suffer. That is the Gospel. So pray for your persecuted brothers and sisters in Christ, for the suffering around the world; because one day you will need them, you will need their voice. I know they will be there praying for you when the time comes.” –VOM contact suffering persecution

domain: sociology


text:
Hotel investments provide opportunity for a good level of regular annual income, which should rise as the asset management efficiencies are generated – leading to capital growth following the increase in profitability.
We currently manage hotels across the UK and Ireland, including the Holiday Inn Express in Folkestone (located seven miles from the Channel Tunnel terminal) and the Marine Hotel in Troon – which hosted the 2016 UK Open Golf Championship.
Working closely with our asset managers, we aim to acquire interests in under-valued and under-performing hotels. These are primarily in key strategic locations outside of London, where opportunities exist to rebrand and/or reposition them – to enhance trading revenues, generate strong annual cash yields, and significantly improve capital value.
Our hotels typically benefit from a franchise agreement with a branded hotel chain – for example Holiday Inn – and are run day-to-day by local staff, overseen by an experienced hotel management company.
Our partners have a proven track record in originating and completing hotel acquisitions in the UK. Together, they provide a comprehensive service which includes deal origination, the provision of debt finance and post-acquisition, as well as the asset management of the hotels. We fund the acquisition of hotels with no more than 50% funding from lending banks – but if investors do not want to participate in this trade, then it can be noted on their Application Form.
A hotel partnership acquires the freehold, or long leasehold, interest in each hotel – with full title at the Land Registry. These type of assets underpin the security of capital afforded by this trade. Our partners have a proven track record in originating and completing hotel acquisitions in the UK and will provide a comprehensive service – including deal origination and the provision of debt finance. Post-acquisition, they will then manage the hotel assets.
Example Investment: Murrayshall Country House Hotel & Golf Club, Perth, Scotland. Acquired November 2016.Download our Investment Sector overview
Meet Our Investment Managers
Charles formed Hetherley Capital Partners in 2012. He has more than 15 years’ experience in hotel real estate investment, finance, hotel operations and development, as well as hotel asset management and business strategy.
As principal, Charles overseas the Hetherley team in all aspects of the business, including deal origination, investment analysis and asset management of the existing portfolio. Charles heads up investor relations for Hetherley and acts as the primary point of contact for all strategic partners of the business. He also takes a leading role in business development, with a particular focus on expanding Hetherley’s investor base through corporate JV partnerships.
Prior to establishing Hetherley, Charles spent 10 years advising private and corporate investors in hotel investment, development and feasibility with TRI Consulting (acquired by CBRE Hotels). In that time, he built up a detailed understanding of the UK and Irish hotel markets, and is now a regular contributor to Pan-European hotel industry conferences – travelling extensively to assess investment opportunities for Hetherley and its capital partners.
Charles received his BSc (Hons) undergraduate degree from Surrey University in 2002. He is married with three children and enjoys most sports that keep him fit and active. He is a regular charitable donor and has undertaken a number of challenges raising money for selected charities, including GAIN, Cancer Research UK and MIND.
Hotel investments are accessible through the following services:
Stellar Business IHT Service
A discretionary managed service which provides numerous succession planning and tax efficiency options for business owners – either following the sale of a company, or to invest excess company capital to maintain tax efficiency.
Stellar Growth IHT Service
This discretionary managed service involves the creation of a privately owned limited company for each investor, to access a portfolio of asset-backed Investment Sectors – targeting 5% growth per annum.


text:
Amaya Leisure to invest $100 million into four new Sri Lanka hotels
Amaya Leisure has revealed that it is looking to invest US $100 million over the next four years into four new Sri Lanka hotels as the company looks to expands to meet the demands of the post-war travel market.
Amaya Leisure, a company connected to the Vallibel group in Sri Lanka, have announced that they will be investing US$100million into four Sri Lanka hotels over the next four years as part of an expansion plan to meet the demands of post-war travellers booking Sri Lanka holidays.
Managing Director of Amaya Leisure, Lalin Samarawickrama, reportedly told a forum in Colombo: "We intend on completing all these projects before 2014."If we (Sri Lanka) are to reach 2,500,000 tourists, we need bedrooms. Amaya intends on expanding to 1,000 bedrooms by 2014. That is our target."
With over 447,000 travellers visiting Sri Lanka last year and an expected 600,000 travellers to visit the island and stay in Sri Lanka hotels and resorts this year, the island country is seeing a welcomed boost in tourism after the end of the 30 year long civil war. In June 2010, tourist arrivals were up by 48 percent after the war ended in May 2009.
Amaya Leisure, a holding of Connaissance Holdings, currently has three Sri Lanka hotels and resorts; Amaya Hills, Kandy, Amaya Reef, Hikkaduwa; and Amaya Lake, Dambulla.
Starting off in 1981 as the tour operator, Connaissance, Amaya Leisure was established just before the three decade-long war began following ethnic riots in 1983.
In 1994, the company built their first chalet-type hotel near Kandalama Lake in central Sri Lanka and in 2005; this was rebranded and refurbished into a four star luxury hotel.
Amaya Leisure is now planning to expand their portfolio with the construction of four new luxury hotels in Sri Lanka. One of the proposed Sri Lanka hotels is Amaya Sands which will be built on the south western coast in Wadduwa and is expected to cost US$15million. Samarawickrama said: "Foreign architects are finalizing the designs. The concept is there and hopefully by 2012 we should be able to put this out in to the market."
The remaining Sri Lanka hotels include a 200 room property set upon 14 acres of land in Negambo and set to cost US$30million; a 30-room chalet-style property on 24 acres of land in Kalpitiya, expected to cost US$15million; and a 200 room luxury resort in Mirissa, near Matara on the south coast, which is expected to cost US$20million. The remaining US$10million is expected to be spent on refurbishing the existing Sri Lanka hotels.
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Enjoy a relaxing stay in an ideal setting surrounded by lush frangipanis, swaying palms and the glistening blue waters of Bentota's beaches.
- Discover Sri Lanka's cultural wonders from a soothing luxury retreat oozing 18th century charm
- Bask in acres of lush gardens framed by frangipani trees and facing a secluded golden sand beach
- Be wowed by the Dine by Design concept, allowing you to eat out in an array of scenic settings
Discover local life in a fishing village, explore rainforests, Temples and much more on Sri Lanka's West Coast, and enjoy great value for money
- All rooms include a balcony or terrace with either Partial Sea View or Ocean View
- Enjoy Yoga, Snooker, access to 2 Swimming Pools & much more
- Access to the Sahana Spa and leave your daily stress behind with the magical touch of various spa treatments
Enjoy a wonderful beachside haven of tranquillity, days of luxury and the chance to see the island's green hills carpeted by lush plantations
- All rooms feature a Balcony or Terrace with spectacular views
- Enjoy the huge selection of activities including Volleyball, Tennis, Squash & more
- Relax at the Ayurveda Wellness & Spa with the best quality services that will make your stay unforgettable
Private tour visiting many great cities such as Sigiriya, Polonnaruwa and Kandy
- The Highlights of Sri Lanka Private Tour showcases some of the most interesting cultural and historic sites in the world
- Enjoy the game drive through Minneriya National Park which is a home to sambar deer, leopards and elephants and features the ancient Minneriya Tank
- English speaking chauffer guide for the entire round tour
Soak up the sunshine that pours down on Sri Lanka's Indian Ocean coastline at Tangerine Beach. Make the scenic resort...
Sri Lanka is the land of endless natural beauty, with ancient sight and cultural treasure, you'll be sure to embark on a...
The Highlights of Sri Lanka and beach Tour showcases some of the most interesting cultural and historic sites in the...
There are few better places from which to enjoy the calming effect of the Indian Ocean breeze than at Avani Kalutara in...
Take a trip to Sri Lanka and enjoy the warm hospitality at Avani Bentota. It is a resort located along the popular...
Discover a world of peace and relaxation at the all-inclusive Mermaid Hotel Club in the gorgeous Kalutura Beach, Sri...

domain: travel


text:
A statement yesterday by the spokesperson of JAMB, Mr. Fabian Benjamin, said the ‘professor’ was apprehended by the monitoring team of the registrar of JAMB in an examination hall at Brix Academy, Jabi, Abuja.
He said, “His identity was shrouded in dubious tales. He claimed during interrogation that he was one of the professors selected by the Board to oversee the conduct of the examination.
“He also claimed that he was awarded a professorial position by Christian University, Isolo, Lagos and that he had two professorial awards from “Becket” University England and Freedom University.
“He similarly claimed to be a staff of Anchor Polytechnic, Kubwa, Abuja, an institution that does not exist. According to him, he said he is the president of the United Nigeria Chaplin Institute of Peace Academy.
“However, it was discovered that he had a daughter who was taking the 2019 UTME in the same centre where he was caught.”
“The Board’s operational guideline does not allow unauthorised persons into the examination hall.
“The Professor was, therefore, not one of those selected by the Board to monitor the conduct of the 2019 UTME,” the statement said.
Police reacts as Vikings, Black Axe cult groups clash in UNIUYO
The Public Relations Officer of the command, SP Odiko MacDon, who confirmed the incident to newsmen in Uyo, said the police were aware of the incident and that normalcy had returned to the campuses.
“The Commissioner of Police is trying to meet with the management of the university to strategise on how to tackle the menace and ensure adequate security of the university,” he said.
A student of the university, who preferred anonymity, lamented incessant attacks from cult groups apparently from outside the varsity campuses.
He said that one student was killed in the afternoon on Tuesday at the main campus, while three students were killed at the permanent site of the university at about 9pm same day.
“Before the last matriculation that held on Wednesday April 17, there were shoot-outs. During the matriculation there were clashes between cult groups.
“Just yesterday a student was shot dead during a clash between two cult groups at the Annex campus.
“I heard that other three students were shot dead last night at the Main Campus at Nwaniba, making it four dead persons at the campuses,” he said.
Also speaking, Mr Boniface Bassey, a students of Faculty of Arts called on the state government to intervene in the development.
He suggested that the school management should bring plain-cloth security personnel to secure the institution.
Bassey further called on management of UNIUYO to fish out perpetrators of the clashes for prosecution.
“Officers with mufti should come into the campuses and work with the school security to fish out the perpetrators,” he said.
Mrs Blossom Okorie, Director of Information, University of Uyo confirmed the incident but said she was not in the position to speak further on the incident.
“We have reported the matter to the police and I cannot comment on matter,” she said.
She said that the Vice Chancellor, Prof. Eniefiok Essien, was the only person to speak on the issue.
Banditry: Buratai issues order to Army commanders
The Chief of Army Staff, Lt Gen. Tukur Buratai has extolled officers and men of the Nigerian Army for the various successes recorded so far in safeguarding Nigeria’s territory.
NAN reports that the Army Chief made the commendation at the opening of the 2019 first quarter Chief of Army Staff Conference at Jaji in Kaduna State on Wednesday.
He however said in spite of the successes, the troops must think out of the box to keep the terrorists on the run.
Buratai said credible intelligence was necessary in meeting various security challenges in the country and directed commanders to work closely with locals, so as to decisively deal with terrorists, bandits and other criminal gangs.
“It is gratifying to note that the NA within the First Quarter 2019 has performed creditably well starting with the seizing of initiatives and turning the battle momentum against the Boko Haram terrorists in the North East.
“And rounding up with the professional manner with which the NA supported the civil authorities, as part of our constitutional role, to ensure the successful conduct of the 2019 General Elections.”
He added: “Despite reports of some alleged cases of infractions by some personnel, which are being investigated by a high level committee, I am elated with the encomiums and accolades showered on the NA for the high level of professionalism exhibited in the conduct of the elections. I say well done to each and every one of you.
“I must also acknowledge the resilience and doggedness of our troops in reversing the ugly trends that almost marred our concerted efforts in the North East,” Buratai said, noting that Boko Haram alliance with the Islamic State West Africa Province (ISWAP) had been dealt with.
“That is why Operation YANCIN TAFKI was launched to effectively ensure the total destruction of the insurgents hibernating and operating from the ‘tumbuns’ with a view to crippling their ability and capability to wage any form of war.
“The operational successes recorded in this First Quarter 2019 in this fight have further underscored the NA’s resolve to defend the territorial integrity of Nigeria.”
On banditry and kidnapping in the North West and North Central Zones, Buratai said new strategies are being adopted to address the ugly trends.
“This has necessitated the launching of operation HARBIN KUNAMA III.
“The concept of this operation will require the combat arms as well as all combat support and combat support services working in synergy with other security agencies, the traditional institutions and other stakeholders to get rid of criminal elements.
“We must ensure information flow that will be converted into actionable and timely intelligence for troops to take proactive measures and to support and drive targeted operations.
“It is important that commanders in the field relate with the society and other security agencies in their efforts to bring the high rate of kidnapping, acts of banditry and attacks on villages leading to killings in Zamfara, Sokoto, Kaduna and Katsina States to an end.
“There must be sustained synergy, cooperation and collaboration with all stakeholders in these endeavours.”
The Army chief urged the media to be patriotic and stop glorifying terrorists and criminals in the country.
“I will seize this opportunity to caution the media and the general public not to glorify bandits, armed robbers, kidnappers and ethnic militias to the extent that they are seen as stronger than the military and other security agencies.
“The criminals and bandits are only as strong as the society paints or view them and further propagated by the media.
“It is important to state that information and intelligence flows from the people and indeed from the press and the public.
“Such criminal activities are intelligence based and the people, are in this regard, to provide timely information and clear identifications of the perpetrators.
“Undue publicity and praise singing of the bandits and other criminal elements would only motivate them to commit more criminal acts at the detriment of the society.
“Therefore, the press, the general public, security analysts and public commentators are advised to show more maturity and due diligence in this collective effort of ridding the country of criminals and criminalities.”
Police In Lagos Nab Two Young Men Fighting Over N30,000 After Stealing Female Pants
The Police in Lagos have arrested two young men for allegedly stealing a female underwear.
According to the information gathered from the Police, the two men are Friday Emmanuel, 21, and Obiora Nwabueze, 29.
The suspects were said to be arrested under the Mile Two Bridge, Lagos, by operatives of the Satellite Division after they had engaged each other in fisticuffs, following the failure of one of the parties to keep to the terms of agreement regarding the stealing of the panties.
Nwabueze had earlier agreed to pay Emmanuel N30,000 on succesful execution of the ‘operation’ — a promise he did not want to redeem after Emmanuel had fulfiled his own part of the deal.
According to a statement by Bala Elkana (DSP), spokesman of the police, when Emmanuel brought the panties, instead of paying him the agreed amount, Nwabueze attempted to take them by force, which and that caused a fight.
“During interrogation, Emmanuel regretted listening to his friend Nwabueze, who was pestering him to get him female underwears he desperately needed to become rich,” Elkana said. “Both suspects were Wednesday charged to Magistrate Court 23, at Mbah Road, Ajegunle, Lagos for conspiracy and stealing.”
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The district includes the neighborhoods of Canoga Park, Reseda, Tarzana, Winnetka, and Woodland Hills.
City Council RepresentativeBob Blumenfield
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Bring the family for a fun event this summer! Moana — Free and open to all.


The Pune Smart City has revolutionized urban safety with the integration of over 2700 live camera feeds into its Integrated Command Control Center (ICCC). This collaborative effort with Pune City Police has significantly improved surveillance and response capabilities, making the city a safer place for its citizens.
A smart city official said, “Pune Smart City, in partnership with Pune City Police, has prioritized safety and sustainable security for its residents. By integrating an extensive network of live camera feeds into the ICCC, the city has established a cutting-edge surveillance system that operates 24/7, enabling real-time monitoring and prompt incident response.”
The vision of Pune Smart City and its dedication to leveraging advanced technology for public safety. The implementation of the ICCC not only enhances vigilance but also strengthens the city’s security infrastructure, positioning Pune as a forward-thinking metropolis that seamlessly blends safety with sustainability.
The comprehensive surveillance initiative undertaken by Pune Smart City, showcasing its commitment to utilizing state-of-the-art technology. By embracing strategic partnerships and harnessing the power of advanced surveillance systems, Pune Smart City sets a new benchmark for urban safety, serving as an exemplary model for cities worldwide.
The widespread interest in Pune Smart City’s ICCC from other smart cities across the country. Highlighting the significance of collaboration and knowledge sharing, city officials discuss their recent hosting of the Lucknow Smart City team at the ICCC, aiming to foster growth and development in smart cities throughout India.


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Between them, Edinburgh’s National Galleries of Scotland and their accompanying collections of over 100,000 artworks house a great number of masterpieces, including greats like Titian, Rubens, Da Vinci, Rembrandt and Botticelli. From the mesmerising to the mad, explore our guide to the top must-see artworks in Edinburgh.
Revd Robert Walker Skating On Duddingston Loch by Sir Henry Raeburn
Renowned for its unique composition and setting, Reverend Robert Walker Skating on Duddingston Loch or The Skating Minister by Scots portrait painter Sir Henry Raeburn features a lone skater performing a sophisticated pose on the loch near Arthur’s Seat. Since 1949, this oil painting, which was painted during the Scottish Enlightenment, has served as one of the most renowned Scottish paintings. Interestingly, 2005 saw an attribution conflict when an art curator suggested the artist was actually Frenchman Henri-Pierre Danloux, although the gallery director believes it is indeed Raeburn.
The Three Ages Of Man by Titian (Tiziano Vecellio)
Titian’s The Three Ages of Man is an allegory for the fleeting nature of life itself. Between a winged cupid and star-crossed lovers, in tandem with the church and old man pontificating over two skulls, there’s a lot to be said about the goings-on in this oil on canvas in The Scottish National Gallery .
The Virgin And Child (‘Bridgewater Madonna’) by Raphael
Although the majority of works by High Renaissance painter and architect Raphael are in the Vatican Palace, The Virgin And Child (‘Bridgewater Madonna’) adorns Edinburgh’s Scottish National Gallery. All eyes rest upon the naturalistic yet synchronized pose between Virgin and Child due to Raphael painting over the initial landscape background.
A Vase Of Flowers by Jean-Baptiste-Siméon Chardin
Art lover or not, it’s impossible not to marvel at the calculated restraint and mastery of simplicity demonstrated by 18th-century painter Chardin in one of his most renowned paintings. Housed in the Scottish National Gallery, this striking artwork is Chardin’s only surviving flower piece. The alluring nature of the light and meticulously balanced composition surrounding the flowers painted by this master of light is quite remarkable.
Robert Burns by Alexander Nasmyth
Housed in the fabulous Gothic revival Scottish National Portrait Gallery amidst a sea of famous Scots faces, is a rather captivating portrait of Scottish Bard Robert Burns. The work of Scottish landscape and portrait painter Alexander Nasmyth, this oil on canvas piece was bequeathed by Colonel William Burns in 1872.
The Family Of John Hay, 1st Marquess Tweeddale by Sir John Baptiste de Medina
Attributed to Flemish-Spanish portrait painter Sir John Baptiste de Medina, this painting of the family of John Hay, the 1st Marquess of Tweeddale and Lord High Chancellor of Scotland, portrays an intriguing glimpse into the personalities of his family members. The expressions on each face capture the moment, inviting the viewer to think what on earth was being discussed!
Princess Elizabeth And Princess Anne, Daughters of Charles, I, 1637 by Sir Anthony van Dyck
Out of the museum’s 3000 paintings and sculptures, in addition with the 25,000 prints and drawings and 38,000 photographs (yes, there are that many), this intimate portrait of Princesses Elizabeth and Anne shows a tender glimpse of them as infants. The glow radiating from the pearls and use of light upon the bunnet is all thanks to the artistic prowess of Flemish Baroque artist painter Sir Anthony van Dyck.
Landform by Charles Jencks
Landform by highly acclaimed American architect and architectural historian Charles Jencks is an unmissable visual tie between the buildings of Modern One and Modern Two. Through combining a mix of garden and land art and sculpture, Jencks provides some food for thought and a stunning precursor to the wondrous potion of works inside both galleries and also the maze of sculptural masterpieces — including the evocative ‘There Will Be No Miracles Here’ sign by Nathan Coley and Domino by Eduardo Paolozzi.
Tragic Form (Skate) 2014 by Ken Currie
The year 2016 saw the arrival of Tragic Form (Skate) 2014 by renowned Scottish artist Ken Currie, a most eye-opening piece and integral component of his artistic journey. This sizeable painting can be located in Modern One and certainly can’t be missed.
Vulcan by Eduardo Paolozzi
Made from welded steel between 1998 and 1999, Vulcan is an enormous, imposing sculpture by Scots sculptor Eduardo Paolozzi, of which the Scottish National Galleries hold many of his works. Vulcan, the Roman God of Fire and Blacksmith for weapons of war, is used by Paolozzi as an archetypal sculptor figure. This half machine, half man monument is a take on the modern era and sculptural narrative about current life and accompanying morals.


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Over the years, Nana Ama McBrown has proven to be a force to reckon with respect to Ghana’s movie industry.
She has for so long been sharing lovely photos and messages with her social media fans. YEN.com.gh brings to you 5 times she took social media by storm:
In this photo, her golden hair, glowing skin, beautiful smile and yellow dress was all she needed to charm her fans.
Nana Ama settled for a regal look in this photo, as she looked beautiful in a mauve-coloured outfit.
Her smile alone in this photo was enough to melt the hearts of her fans.
Yet another photo of Nana Ama McBrown that literally set social media on fire.
She also shared this photo in which she had a lovely pose for her fans
Ghana Trends: How Anas Aremeyaw Anas caused a Stir With Latest Investigation | Yen.com.gh:
YEN is building a platform where Ghanaians can share local news and own experiences with each other. Witnessing an incident? Want to tell about a local problem? Know someone who is extremely talented and needs recognition? Your stories and photos are always welcome. Send us a message via Instagram or on YEN’s official Facebook page.

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A determined stray kitten who somehow got stuck down a storm drain was taken to safety after rescuers heard his cries.
Video shows the tiny, scared kitten inside the debris-filled drain in Greece.
Read: Cops Use Man's Cat to Coax Him Down From Ledge After 3-Hour Standoff
See photos of the tiny little kitten:
When rescuers arrived to help the terrified cat, he wouldn't stand still long enough for them to get him out so they covered a scarf with cat food. As he gratefully ate the food, they were able to scoop him out to safety.
He was fed by his rescuers and returned to his cat colony. Stray cats are a huge problem in Greece so the Second Chance Animal Rescue Society is working to spay and neuter as many as they can.
Also see these other adorable felines:

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Allow me to introduce myself to those of you that do not know me yet! I’m Carl Hallowell. I specialize in TRADITIONAL JAPANESE TATTOOING and work by APPOINTMENT ONLY out of my PRIVATE STUDIO ROOM at Heart in Hand.
2020 marks 25 YEARS OF TATTOOING for me! But my learning continues! I am a student of MASTER SHODAI HORIKOI of Toyohashi Japan, and a member of the HORIKOI FAMILY. I am proud to offer you the authentic work that this connection allows me the privilege to present.
My honorific Japanese title is “HORISHO”. “Sho” means “Straight, Right idea”. This suits me well, as I believe that the idea itself is the most important part of every tattoo. I cherish my clients, and work hard to help each of them to arrive at the “right idea” for their work.
I am now practicing TEBORI, the Traditional method of JAPANESE HAND TATTOOING. An old adage says, “A western tattoo is at its best when it is first completed, a Japanese tattoo gets better with age.” This is in large part due to the tebori method. Although I still outline with the machine, and am still available for machine tattooing, I am proud to offer this very rare service to America.
The Japanese tattoo is perhaps the most recognizable tattoo in the world; it’s rich traditions have been perfected by many a JAPANESE TATTOO MASTER over the years. It is the originator of the SLEEVE tattoo, the BACKPIECE tattoo, and the FULL BODYSUIT tattoo. The Japanese tattoo is based on the human body itself. This makes it perhaps the most powerful art form humanity has ever known.
I would love to share the Japanese tattoo with you. It is a very deep and complex form. The stories and motifs must be in HARMONY, the designs and their placements must be CORRECT, and the rules of the form must be followed. It would be my honor to help you achieve each of these unique attributes of the JAPANESE TRADITIONAL TATTOO!
Please CONTACT ME at email@example.com for a consultation and appointment. I personally respond to all emails within 3-4 days. My schedule books several months in advance, so I apologize for the delay. But I promise you it will be worth the wait! THANK YOU!


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Sarah Burton opens new chapter for Alexander McQueen
The storytelling of Sarah Burton for the house Alexander McQueen has always been deeply rooted in British heritage. Since the beginning of men’s collections back in 2005, they had been shown in Milan, and then in London for a few seasons. Since then, the menswear collection has been absent from the catwalk, favouring instead presentations and sophisticated lookbooks.
This year, for the first time, Sarah Burton showcased her Alexander McQueen spring/summer 2018 collection in a runway format for the Paris Menswear Fashion Week. On the invitation card was the poem The Seasons: Spring written by James Thomson in 1728 for the Countess of Hertford. The poem was separated in a block structure made of red threads printed on tracing paper. The fashion show was presented in the orangerie of the French Senate in the grounds of the Luxembourg Gardens in Paris.
The late designer Alexander McQueen built a solid reputation in the fashion industry as a tailoring expert. He worked at Anderson & Sheppard, and then at Gieves & Hawkes, two of the biggest bespoke tailoring houses in London. Burton pursues his heritage in delivering sharp garments and went far beyond classical tailoring.
The show started with a clean double-breasted coat, 6 on 2 buttons, with peak lapels of black fabric. The second look was a double-breasted jacket with the same cut and details. On the third look, we saw a sleeveless stiff bib-fronted dress shirt in ivory leather. Black trousers were slim fitted with a large broad leather ivory or scarlet red stripe on the side.
Later, Burton played with different types of fabrics such as bonded leather, washed leather, silk poplin, knitwear, selvedge Japanese denim, broderie anglaise, wool silk jacquard while maintaining precision of tailoring. The colour palette was black, beige, white, ivory and a great touch of scarlet and blood red.
Destructuring the basics of British tailoring, the oversized cotton garbadine trench coat was reversed inside out, revealing wool silk jacquard patchwork of a subtle guncheck. Suits made of patchwork Prince of Wales check matched perfectly, constituting a technical feat.
Then came a more dreamy silhouette: oversized shirts edged in broderie anglaise worn with pajamas trousers, chunky knitwear, selvedge Japanese denim with orienteering maps and drawings of Rudyard Kipling’s poem The Explorercompleted with red threads.
After the exploration, the Alexander McQueen man might have found his way in the maze. He was at peace with nature. His suit and coat were embroidered with colourful flowers.
The raw beauty of wool silk woven jacquard is used inside out. The soft part is worn close to the skin, and loose threads of silk and wool gave an amazing complex structure to tuxedo jackets and coats. The last look, a stunning black coat with peak lapels, embroidered with a glittering “Tree of Life”, was a technical masterpiece.
Without a doubt, Burton opened a new chapter for Alexander McQueen. Her poetic storytelling, subtle British touch, tailoring expertise, and flora and fauna embroidery resulted in a dreamy collection with elegant silhouettes.
Burton’s poetic storytelling, subtle British touch and tailoring expertise results in a dreamy collection with elegant silhouettes


A Lakefield woman was injured early Monday as a result of a car-vs.-deer crash on Interstate 90.
According to the Minnesota State Patrol, a 2021 Chevrolet Trailblazer operated by Kerry Ellen Schmidt, 50, of Lakefield, was westbound on I-90 when it struck a deer. The incident took place at 6:30 a.m. Monday near the Adrian exit at milepost 26.
Schmidt was transported to Sanford Worthington Medical Center with a non-life-threatening injury. The Nobles County Sheriff’s Office and Sanford Worthington Ambulance assisted at the scene.


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Apoptosis (2012) 17:90–101 DOI 10.1007/s10495-011-0658-1
Mammalian target of rapamycin regulates isoliquiritigenininduced autophagic and apoptotic cell death in adenoid cystic carcinoma cells Gang Chen • Xiang Hu • Wei Zhang • Ning Xu • Feng-Qin Wang • Jun Jia • Wen-Feng Zhang • Zhi-Jun Sun • Yi-Fang Zhao
Published online: 29 September 2011 Ó Springer Science+Business Media, LLC 2011
Abstract Previous studies, including those from our laboratory, have demonstrated that isoliquiritigenin (ISL), a flavonoid isolated from licorice, is a promising cancer chemotherapeutic agent. However the mechanisms underlying its anticancer effects are still far from clear. We now show, for the first time, that ISL triggers the mammalian target of rapamycin (mTOR)-dependent autophagic and apoptotic cell death in adenoid cystic carcinoma (ACC). Exposure of both ACC-2 and ACC-M cells to ISL resulted in several specific features for autophagy, including the appearance of membranous vacuoles, formation of acidic vesicular organelles, punctate pattern of LC3 immunostaining, and an increase in autophagic flux. Moreover, ISL treatment also resulted in significantly increased apoptosis in ACC cells. The ISL-mediated autophagic and apoptotic cell death were obviously attenuated by transfection with dominant negative Atg5 (DN-Atg5K130R) plasmids or treatment with 3-methyladenine(3-MA). In additon, the data also revealed that the autophagic and apoptotic cell
Gang Chen and Xiang Hu contributed equally to this article.
Electronic supplementary material The online version of this article (doi:10.1007/s10495-011-0658-1) contains supplementary material, which is available to authorized users. G. Chen X. Hu W. Zhang N. Xu F.-Q. Wang J. Jia W.-F. Zhang Z.-J. Sun (&) Y.-F. Zhao (&) The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory of Oral Biomedicine Ministry of Education, Department of Oral and Maxillofacial Surgery, School & Hospital of Stomatology, Wuhan University, Wuhan, China e-mail: [email protected]
Y.-F. Zhao e-mail: [email protected]
death induced by ISL occurred through a mTOR-dependent pathway. More importantly, the xenograft model using ACC-M cells provided further evidence of the occurrence of ISL-induced autophagy and apoptosis in vivo, correlating with the suppresson of mTOR activation as well as up-regulation of Atg5 expression. Taken together, these findings in our study suggest that induction of mTORdependent autophagic and apoptotic cell death may be an important mechanism in cancer chemotherapy by ISL. Keywords Isoliquiritigenin Adenoid cystic carcinoma Autophagy Apoptosis mTOR
Introduction Adenoid cystic carcinoma (ACC) is one of the most common malignancies of the major and minor salivary glands [1, 2], and accounts for approximately 15–25% of all the carcinomas at these locations . This neoplasm often presents a prolonged clinical course , providing a large window of opportunity for intervention to prevent or slow its malignant progression. Thus, identification and preclinical/clinical development of novel agents that are nontoxic to humans but can delay the onset and/or progression of ACC are highly desirable. Epidemiological studies have shown that dietary intake of flavonoid-containing fruits, vegetables and beverages is associated with a low risk of cancer [5, 6]. Isoliquiritigenin (ISL), 20 ,40 ,40 -3-hydroxychalcone [7, 8], is a natural flavonoid that has been shown to be nontoxic to humans but that has various biological properties, such as antiinflammatory, antioxidant, and antiplatelet aggregation, as well as vasorelaxant and estrogenic effects [8, 9]. More importantly, ISL has also been demonstrated to possess
Apoptosis (2012) 17:90–101
significant antitumor activities, including inhibition of proliferation and/or induction of apoptosis  as well as prevention of metastasis [11, 12]. Nevertheless, the precise mechanisms underlying the antitumor activities of ISL are yet to be elucidated. Our recent study  revealed that ISL treatment suppressed the growth and angiogenesis-induction activity of ACC cells in vitro and in vivo. We found that treatment with ISL (0–20 lM) for 24 h could inhibit the growth of ACC cells in cultures without the occurrence of cell death. Meanwhile, we also observed that when the treatment period was extended to 48 or 72 h, cell viability was significantly reduced by the same concentrations of ISL. However, the mechanisms behind the cytotoxicity exerted by ISL have not been fully elucidated. The only known intracellular responses are the suppression of the mTOR pathway correlating with concurrent activation of c-Jun NH2-terminal kinase (JNK), and the inhibition of extracellular signal-regulated kinase (ERK). Of note, the mTOR signaling pathway has assumed a central role in the regulation of autophagy, which is an evolutionarily conserved and dynamic process for bulk degradation of cellular macromolecules and organelles [13, 14]. The present study is designed to investigate whether ISL-induced mTOR suppression may lead to autophagy in ACC cells, and whether the occurrence of autophagy may contribute to the cytotoxicity exerted by ISL. Here, we initially evaluated the effects of ISL in cultured ACC cells under cytotoxic conditions (0–20 lM for 48 h), and found that under these conditions ISL treatment not only induced autophagy in ACC cells, but also resulted in significantly increased cell apoptosis. This related to the up-regulation of mTOR-dependent Atg5 expression. Further, we assessed the in vivo effects of ISL by establishing a xenograft model using ACC-M cells, and also observed the mTOR-dependent autophagic and apoptotic cell death induced by ISL. Taken together, these findings suggest that ISL can induce mTOR-dependent autophagic and apoptotic cell death in ACC cells, which likely serve as an important mechanism in its cancer chemotherapeutic effects.
against b-actin was purchased from Santa Cruz. The expression vectors encoding constitutively active mTOR (pRK5-CA-mTOR), as well as the corresponding empty vectors (pRK5) were kindly provided by Dr. Fuyuhiko Tamanoi (University of California, USA). The expression vectors encoding dominant negative Atg5 mutant (pCIneo-DN-Atg5K130R), as well as the corresponding empty vectors (pCI-neo) were provided by Prof. Tamotsu Yoshimori (National Institute for Basic Biology, Okazaki, Japan).
Materials and methods
Detection of acidic vesicular organelles
Chemicals and antibodies
The formation of yellow-orange acidic vesicular organelles (AVOs) was detected as we described earlier . Briefly, both ACC-2 and ACC-M cells (2 9 105) were grown on coverslips in 6-cm dishes and allowed to attach by overnight incubation. After treatment with increased concentrations of ISL for 48 h, cells were stained with 1 lg/ml acridine orange (AO) in PBS for 15 min, washed with PBS, and then examined under a Leica fluorescence microscope.
Unless otherwise noted, all chemicals and reagents including ISL were purchased from Sigma–Aldrich. Primary antibodies for phospho-mTOR (Ser2448), mTOR, phospho-S6 (Ser235/236), S6, Atg5, LC3, Bax, Bcl-2, and cleaved-poly (ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology. Primary antibody
Cell culture and transient transfection The low (ACC-2) and high (ACC-M) metastatic cell lines of human salivary ACC were obtained from the China Center for Type Culture Collection, and were grown in DMEM medium supplemented with 10% FBS. The cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C. For transient transfection, ACC cells were seeded in 6 cm culture dishes at a density of 106 cells/dish, and then transfected with CA-mTOR, DN-Atg5K130R as well as corresponding vector plasmids using the LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s instructions. The expression levels of p-mTOR and Atg5– Atg12 complex after transfection were determined by Western blotting. Transmission electron microscopy The transmission electron microscopy was performed according to our previous procedures . Briefly, both ACC-2 and ACC-M cells were incubated with either DMSO (Control) or 20 lM ISL for 48 h. Cells were then fixed with 2.5% electron microscopy grade glutaraldehyde, postfixed in 1% osmium tetroxide with 0.1% potassium ferricyanide, dehydrated through a graded series of ethanol (30–90%), and embedded in Epon. Ultrathin sections (65 nm) were cut, stained with 2% uranyl acetate and then examined using a Hitachi H-600 Transmission Electron Microscope.
Immunofluorescence microscopy for LC3 localization The immunofluorescence for LC3 localization was performed as previously described . Briefly, both ACC-2 and ACC-M cells (2 9 105) were plated on coverslips in 6-cm dishes and allowed to attach by overnight incubation. Cells were then treated with increased concentrations of ISL for 48 h. After that, the cells were washed with PBS and fixed in 4% paraformaldehyde overnight at 4°C. They were then permeabilized with 0.1% Triton X-100 for 10 min and blocked with bovine serum albumin (BSA) buffer for 1 h. Cells were then incubated with the anti-LC3 antibody (1:50) overnight at 4°C, then treated with fluorescein isothiocyanate-conjugated (FITC) donkey antirabbit antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h. The cells with a punctate pattern of LC3 localization were then visualized and captured using a Leica fluorescence microscope. Determination of apoptosis ISL-induced apoptosis in ACC-2 and ACC-M cells was detected according to our previous procedures [16, 17] as follows: (a) morphological evaluation by Hoechst staining; (b) quantification of cytoplasmic histone-associated DNA fragments with Cell Death Detection ELISAPLUS assay; (c) caspase-3 activity measurement by Colorimetric Caspase Activity Assay kit; and (d) Western blot analysis for Bax/Bcl-2 ratio and PARP cleavage. Western blot analysis Western blot analysis was performed according to our previous procedures [8, 16, 17]. Briefly, ACC cells were lysed, and the total protein was separated using 10% SDS– polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore). The blots were then blocked with 5% non-fat dry milk at room temperature for 1 h, and incubated overnight at 4°C with the corresponding primary antibodies at dilutions recommended by the suppliers, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Pierce) for 1 h. Blots were then developed by a SuperEnhanced chemiluminescence detection kit (Applygen Technologies Inc.). Nude mice xenografts Female BALB/c nude mice (18–20 g; 6–8 weeks of age) were purchased from the Experimental Animal Center of Wuhan University in a pressurized ventilated cage according to institutional regulations. All studies were approved and supervised by the Animal Care and Use
Apoptosis (2012) 17:90–101
Committee of Wuhan University. Exponentially growing ACC-M cells (2 9 106 in 0.2 ml medium) were inoculated subcutaneously into the flank of the mice. After 7 days, tumor-bearing mice were randomly divided into three groups, which were treated with ISL (0.5 g/kg or 1 g/kg p.o. daily; n = 8) or corn oil (Control, 100 ll, p.o., daily; n = 8) for 30 consecutive days. Tumor growth was determined by measuring the size of the tumors daily. Tumor volumes were calculated according to the formula (width2 9 length)/2. The mice were euthanized at Day 30, and the tumors were captured, photographed and embedded in paraffin or frozen at -80°C for the following immunohistochemical analysis as well as for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Statistical analysis All data represent mean ± SEM of three independent experiments. Statistical analysis was performed by Student’s t-test at a significance level of P \ 0.05.
Results ISL treatment activates autophagy in ACC cells As seen in Fig. 1a, treatment of both ACC-2 and ACC-M cells with ISL (20 lM) resulted in typical appearance of membranous vacuoles resembling autophagosomes (double arrowheads). On the other hand, the DMSO-treated control cells exhibited normal healthy looking mitochondria (single arrowhead). Moreover, we also examined the autophagic response of ACC cells to ISL by analysis of LC3 processing and recruitment as well as formation of yelloworange AVOs, which are hallmarks of autophagy. We found that exposure to ISL led to an obvious punctate pattern of LC3 immunostaining in both ACC-2 and ACCM cells, which was rare in the control cells (Fig. 1b). In addition, the data also revealed that ACC cells treated with DMSO primarily exhibited green fluorescence. However, treatment with ISL resulted in formation of yellow-orange AVOs in both ACC-2 and ACC-M cells in a concentrationdependent manner (Fig. 1c). Furthermore, ISL treatment also induced the cleavage of LC3 (an increase in the LC3II/LC3-I ratio) in both ACC-2 and ACC-M cells, and meanwhile up-regulated the expression level of Atg5, an essential protein in autophagosome formation (Fig. 1d). To further distinguish whether ISL-mediated autophagosome accumulation is due to autophagy induction or rather to a block in downstream steps, we performed a LC3 turnover assay by Western blot analysis using the lysosomal inhibitor Bafilomycin A1 to measure the effect of ISL on
Apoptosis (2012) 17:90–101
autophagic flux. The results demonstrated that the difference in LC3-II levels in the presence and absence of Bafilomycin A1 is much larger in case of treatment with ISL when compared to the untreated controls, indicating that autophagic flux is indeed increased by ISL treatment (Fig. 1e). Collectively, these observations strongly suggest that ISL could cause autophagy in ACC cells. ISL treatment also induces apoptosis in ACC cells As shown in Fig. 2a, after treatment with 20 lM ISL for 48 h, several apoptotic morphologic features such as
apoptotic bodies, cell shrinkage and chromatin condensation were observed in ACC-2 and ACC-M cells by Hoechst 33258 staining assay. We also quantitatively assessed the DNA fragmentation in ISL-treated ACC cells using the Cell Death Detection ELISAPLUS kit. Likewise, the results revealed that treatment with ISL resulted in formation of DNA fragments in both ACC-2 and ACC-M cells in a concentration-dependent manner (Fig. 2b). To further verify the ISL-induced apoptosis in ACC cells, we detected the expression levels of some key apoptosis-related proteins in ACC cells treated with ISL. As the results show, the activity of caspase-3 was found to increase significantly
Fig. 1 ISL treatment causes autophagy in ACC cells. Both ACC-2 and ACC-M cells were grown on coverslips in 6-cm dishes and allowed to attach by overnight incubation. Cells were then treated with indicated concentrations of ISL for 48 h. a Detection and quantification for ISL-induced autophagosomes by transmission electron microscopy. b Detection for ISL-induced punctate pattern of LC3 immunostaining. c Detection for ISL-induced formation of yellow-orange AVOs. d The expression levels of Atg5–Atg12 complex and LC3 were detected by Western blotting. e The difference in LC3 expression levels between control and ISL-treated samples with or without Bafilomycin A1 was compared by Western blotting. All data are presented as mean ± SEM from three different experiments with duplicate. ** P \ 0.01 versus the control group
in ISL-treated ACC cells (Fig. 2c). On the other hand, both the ratio of Bax/Bcl-2 and the cleavage of PARP in both ACC-2 and ACC-M cells were significantly increased by ISL in a concentration-dependent manner (Fig. 2d), thus confirming the apoptosis-induction by ISL. Inactivation of Atg5 prevented ISL-induced autophagy in ACC cells As described above, Atg5 is an essential protein for autophagosome formation, and Atg5-deficience can significantly diminish the number of autophagic vesicles [18, 19]. Here we evaluated the role of Atg5 in ISL-induced autophagy in ACC cells by transient transfection of DN-Atg5K130R prior to ISL treatment. DN-Atg5K130R is a dominant negative mutant of Atg5, in which Lys130 of Atg5 was replaced with Arg, and has been previously shown to inhibit its conjugation with Atg12 and suppress vacuole formation . Under normal conditions, the conjugation between Atg5 and Atg12 was formed via an isopeptide bond between the C-terminal glycine of Atg12 and Lys130 of Atg5. While when the Lys130 of Atg5 was changed to Arg, the conjugation between Atg5 and Atg12
Fig. 2 ISL induces apoptosis in ACC cells. Both ACC-2 and ACC-M cells were treated with indicated concentrations of ISL for 48 h. a The morphologic changes were captured using fluorescence microscopy with Hoechst 33258 staining. b DNA fragmentation was quantified using Cell Death Detection ELISAPLUS assay. c Caspase-3 activity was measured by Colorimetric Caspase Activity Assay. d The expression levels of cleaved-PARP, Bcl-2 and Bax were determined by Western blotting. All values were presented as means ± SEM from three different experiments with duplicate. ** P \ 0.01 versus the control group
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was destroyed, and therefore the formation of Atg5–Atg12 complex was blocked . As our results show, the punctate pattern of LC3 immunostaining that resulted from ISL treatment for 48 h was significantly reduced by transient transfection of DN-Atg5K130R (Fig. 3a), correlating with the obviously decreased expression of the Atg5– Atg12 complex (Fig. 3b). Consistent with these finding, the autophagic flux induced by ISL in both ACC-2 and ACC-M cells was effectively prevented by inhibition of Atg5 as well (Fig. 3b). Moreover, 3-MA, a well known autophagy inhibitor which could decrease the expression of Atg5 [22, 23], also significantly attenuated the ISL-mediated punctate pattern of LC3 immunostaining (Fig. 3a). All of the above findings indicate that ISL-induced autophagy in ACC cells was dependent on Atg5. ISL-mediated autophagy in ACC cells was a form of cell death but not a protective mechanism The role of autophagy in cancer development and therapy is still unclear. Some studies have suggested that autophagy may serve as a protective mechanism against therapyinduced cell death in various cancer cells [24, 25]. On the
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Fig. 3 Inactivation of Atg5 prevents ISL-induced autophagy in ACC cells. Both ACC-2 and ACC-M cells were transfected with DN-Atg5K130R or pretreated with 3-MA (4 mM), followed by exposure to ISL (20 lM) for 48 h. a Detection and quantification for punctate pattern of LC3 immunostaining. b The protein expression level of the Atg5–Atg12 complex in ISL-treated ACC cells after transfection with DN-Atg5K130R was evaluated by Western blotting. In addition, the difference in LC3 expression levels between samples with and without Bafilomycin A1 was compared by Western blotting. All data are presented as mean ± SEM from three different experiments with duplicate. ** P \ 0.01 versus the control group
other hand, there are also some studies stating that autophagy could be a form of cell death for various anticancer agents [26, 27]. In order to determine the precise role of autophagy in ISL-treated ACC cells, the effect of 3-MA on ISL-induced cell viability reduction was initially evaluated. As shown in Fig. 4a, the decreased cell viability that resulted from 48 h exposure to ISL was effectively rescued by pretreatment with 3-MA, suggesting that ISLmediated autophagy in ACC cells, at least at this exposure time (48 h) and these concentrations (5–20 lM), might be a form of cell death but not a protective mechanism. More importantly, genetic inhibition of ISL-mediated autophagy by transfection with DN-Atg5K130R also significantly prevented the cytotoxicity exerted by ISL in both ACC-2 and ACC-M cells (Fig. 4a), which further validated our hypothesis. After confirming the detrimental role of ISL-mediated autophagy in ACC cells, we further proceeded to explore the relationship between ISL-induced autophagy and apoptosis by examining the effects of 3-MA treatment and DN-Atg5K130R transfection on ISL-induced apoptotic cell death. As the results show, both 3-MA treatment and DN-Atg5K130R transfection significantly but not completely reversed ISL-induced apoptosis in ACC cells, as evidenced by the Hoechst staining (Fig. 4b) as well as by DNA
fragmentation detection (Fig. 4c). Furthermore, pretreatment with 3-MA or transfection with DN-Atg5K130R effectively, and also partially, prevented ISL-mediated PARP cleavage in both ACC-2 and ACC-M cells (Fig. 4d). Together, these results not only demonstrate an essential role of Atg5 in regulation of ISL-mediated autophagy and apoptosis in ACC cells, but also provide additional evidence to indicate that ISL-induced autophagy and apoptosis are interrelated. ISL-mediated autophagy and apoptosis correlated with suppression of mTOR activation Our previous study has shown that ISL treatment for 24 h inhibited the activation of mTOR, a protein kinase which emerged as a key negative regulator of autophagy . In addition, previous studies including those from our laboratory have proven that mTOR was also a potential regulator of apoptosis in various cancer cells [28–30], including ACC cells . Here, we again confirm that treatment with ISL for 48 h could more effectively suppress the activation of mTOR in both ACC-2 and ACC-M cells (Fig. 5a). Meanwhile, the expression level of phosphorylated-S6, a key downstream target of mTOR , was also decreased by ISL in both ACC-2 and ACC-M cells (Fig. 5a). All of
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Fig. 4 Inhibition of autophagy prevents ISL-mediated viability reduction and apoptosis induction in ACC cells. Both ACC-2 and ACC-M cells were transfected without (Control) or with DN-Atg5K130R or corresponding vector plasmids, or were pretreated with 3-MA (4 mM), followed by exposure to ISL (20 lM) for 48 h. a Cell viability was measured using the Vi-CELL cell viability analyzer. b Then morphologic changes were captured using fluorescence microscopy with Hoechst 33258 staining. c DNA fragmentation was quantified using Cell Death Detection ELISAPLUS assay. d The expression level of cleavedPARP was determined by Western blotting. All values were presented as means ± SEM from three different experiments with duplicates. * P \ 0.05 versus the corresponding group without ISL treatment, and # P \ 0.05 versus the control group with only ISL treatment but without any other pretreatment
this evidence suggests that ISL-mediated autophagy and apoptosis in ACC cells is probably due to the suppression of mTOR. To further determine the functional significance of mTOR suppression in the autophagic and apoptotic responses of ACC cells to ISL, we assessed the effect of CA-mTOR over-expression on ISL-mediated autophagy and apoptosis. As evidenced in Fig. 5a, transient transfection with CA-mTOR nearly blocked ISL-mediated mTOR inactivation, but only partially retrieved the up-regulation of Atg5 and the cleavage of LC3 and PARP by ISL. Consistent with these findings, over-expression of CAmTOR in ACC cells significantly but only partially prevented ISL-mediated recruitment of LC3 immunostaining (Fig. 5b). Similarly, the apoptotic ACC cells induced by
ISL, demonstrated by Hoechst staining as well as DNA fragment detection, was also significantly but partially reversed by CA-mTOR transfection (Fig. 5c). These results confirmed that suppression of the mTOR signaling pathway is indispensable for ISL-mediated autophagy and apoptosis, and also indicated that there must be other mechanisms involved, although this requires further investigation. ISL inhibited ACC xenograft growth in association with induced autophagy and apoptosis To further authenticate the effects of ISL described above, animal experiment were designed to test whether ISL could induce autophagic and apoptotic cell death in vivo., and ACC xenografts in nude mice were established using
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Fig. 5 mTOR regulates ISLinduced autophagic and apoptotic cell death in ACC cells. Both ACC-2 and ACC-M cells were transfected with or without CA-mTOR, followed by exposure to indicated concentrations of ISL for 48 h. a The expression levels of p-mTOR, mTOR, p-S6, S6, Atg5–Atg12 complex, LC3 and cleaved-PARP were determined by Western blotting. b Detection and quantification for the punctate pattern of LC3 immunostaining. c The morphologic changes were captured using fluorescence microscopy with Hoechst 33258 staining, and meanwhile the DNA fragmentation was quantified using Cell Death Detection ELISAPLUS assay. * P \ 0.05 versus the corresponding group without ISL treatment, and # P \ 0.05 versus the control group with only ISL treatment but without any other pretreatment
ACC-M cells. As the results show, the growth of ACC-M tumors was significantly inhibited by ISL treatment in a dose-dependent manner compared with the vehicle controls, but the body weight was not affected (Fig. 6a). Importantly, the tumors from ISL-treated mice exhibited significantly increased expression of LC3 (Fig. 6b), suggesting activated autophagic responses. Moreover, treatment with ISL significantly induced cell apoptosis in tumor tissues as evidenced by the TUNEL assay, which is related to the suppression of mTOR activation as well as to the up-regulation of Atg5 expression (Fig. 6b). These results
indicated that ISL could induce mTOR-dependent autophagic and apoptotic cell death in ACC cells in vivo, which might be an important mechanism underlying the antitumor activities of ISL.
Discussion Our previous study has shown that the dietary cancer chemotherapeutic agent ISL could suppress tumor-induced angiogenesis in ACC in vitro and in vivo associating with
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Fig. 6 ISL induced mTORdependent autophagic and apoptotic cell death in vivo. a Tumor volume and body weight of animal in vehicletreated control mice and ISL-treated mice. b Immunohistochemical analysis of the indicated biomarkers in both control and ISL-treated ACC-M tumor tissues. * P \ 0.05 versus the control group
inhibition of mTOR , a highly conserved serine/threonine kinase plays a central role in regulation of autophagy [13, 32, 33]. Thus, the present study was designed to investigate the effects of ISL on autophagy. We exposed ACC cells to increased concentrations of ISL for 48 h, a time period which was enough to cause cell death in ACC cells, and found several specific features for autophagy, including the appearance of membranous vacuoles, formation of acidic vesicular organelles, recruitment of the microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, and an increase in autophagic flux. At the same time, the induction of apoptosis by ISL was also detected, manifested in the formation of DNA fragments, cleaved-PARP and an increased in the Bax/Bcl-2 ratio. Furthermore, both ISL-mediated autophagy and apoptosis were obviously attenuated by treatment with 3-MA or transfection with DN-Atg5K130R. More importantly, the xenograft model provided further evidence for the occurrence of ISL-induced autophagy and apoptosis in vivo, correlating with the suppression of mTOR and up-regulation of Atg5. Taken together, these results strongly suggest that ISL can induce autophagic and apoptotic cell death in ACC cells, which likely serve as an important mechanism in its cancer chemotherapeutic effects. Although the molecular mechanisms for autophagy induction in mammalian cells are still poorly understood, at least 30 autophagy-related genes have been identified in yeast , among which mTOR has emerged as a key negative regulator of autophagy in yeast and possibly in mammalian cells [31, 35–37]. To determine the precise functional significance of mTOR suppresson in ISLinduced autophagy, we evaluate the influence of
CA-mTOR on ISL-mediated autophagic and apoptotic cell death in ACC cells. As the results show, transient transfection of ACC cells with CA-mTOR significantly but only partially reversed ISL-induced autophagy as well as apoptosis, suggesting that there must be other mechanisms involved in ISL-mediated autophagic response, which still needs further investigation. The role of autophagy in cancer development and therapy has been debated with regard to whether autophagy is beneficial for survival or is detrimental for death. There is no doubt that apoptosis, the type I programmed cell death, is a cellular control mechanism for death [16, 38]. However, manipulation of the type II programmed cell death, autophagy, may play dual and opposing roles in cancer therapy [25, 39, 40]. After some anticancer treatments, cancer cells may adapt to survive through activation of autophagy , whereas in other cases the cancer cells might undergo autophagic cell death [42, 43]. In the present study, our results indicated that ISL-induced autophagy at 48 h was indeed detrimental for death, since inhibition of autophagy by 3-MA treatment or DNAtg5K130R transfection significantly reversed ISL-induced reduction of ACC cell viability. Additionally, we found it particularly interesting that short exposure to ISL (12–24 h) could also slightly activate autophagy in ACC cells but showed no effect on cell viability (data not shown). These findings may support a more rational belief that autophagy could facilitate the removal of damaged proteins and organelles, and meanwhile maintain energy metabolism and macromolecular synthesis to promote cell survival under unfavorable stresses; however, if excess or prolonged, autophagy may reversely lead to cell death.
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To date, the relationship between autophagic and apoptotic cell death is also poorly understood, especially concerning whether autophagy directly executes cell death or is the secondary effect of apoptosis. Here our data revealed that ISL-induced apoptotic cell death was both autophagy-dependent and autophagy-independent, because autophagy suppression by DN-Atg5K130R transfection significantly but only partially prevented ISL-mediated apoptotic DNA fragmentation in ACC cells. Indeed, previous studies have shown that enhanced expression of Atg5 can not only promote autophagy but can also increase susceptibility toward apoptotic stimuli irrespective of the cell type [43–45]. These results not only demonstrated the essential role of Atg5 in regulation of ISL-mediated autophagy and apoptosis in ACC cells, but also provide additional evidence to indicate that ISL-induced autophagy and apoptosis are interrelated. In addition to Atg5, there several other signal transduction pathways can elicit both autophagy and apoptosis. The transcription factor p53 is one such molecular. Generally, p53 is known as a quintessential tumor suppressor and apoptosis inducer. However, accumulating evidence suggests p53 also has a positive role in autophagy activation. Moreover, previous study has demonstrated that ISL could induce apoptosis in HepG2 cells through a p53dependent pathway . Thus, we also tried to detect the effects of ISL on p53 signaling pathway in ACC cells, however, unfortunately, we found that ISL treatment failed to affect either the total expression or the transcription activity of p53 (data not shown), suggesting that ISLinduced autophagy and apoptosis is independent on p53 signaling pathway. We then proceeded to test the possible involvement of another molecule Bax in the ISL-induced autophagy and apoptosis, using Bax-inhibiting peptide-V5. As the consequence, inhibition of Bax nearly abrogated ISL-mediated apoptosis-induction in ACC cells (Supplementary Fig. 1a), and meanwhile significantly, albeit partially, prevented ISL-induced viability-reduction (Supplementary Fig. 1c), but showed no effect on ISL-induced autophagy (Supplementary Fig. 1b). More important, we also found that when V5 and 3-MA were used in combination, the cell viability-reduced by ISL could be more obviously prevented (Supplementary Fig. 1c), suggesting that ISL-induced autophagy which is independent on Bax may indeed contribute to part of the cell death in ACC cells. These findings indicated that Bax is indispensable for ISL-mediated apoptosis, but not autophagy in ACC cells; in other words, Bax is only partially essential for ISLmediated cell death. Nevertheless, the precise molecular events, especially those cross talk mechanisms behind the autophagic and apoptotic cell death induced by ISL in ACC cells still needs further investigation.
In conclusion, our results revealed for the first time that ISL could induce both autophagic and apoptotic cell death in ACC cells, relating to the up-regulation of mTORdependent Atg5, which likely serves as an important mechanism in cancer chemotherapy by ISL. Acknowledgments This work was supported by grants from National Natural Science Foundation of China (81072203) to Dr. Z. J. Sun (30801305) to Dr. J. Jia (30973329) to Prof. W. F. Zhang, and (30872894, 30973330) to Prof. Y. F. Zhao. We would like to thank Shelagh Powers in Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research for expert editorial assistance. Conflict of interest disclosed.
No potential conflicts of interest were
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