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Peter Garland – After the WarsPosted by Paul Muller in CD Review, minimalism, Piano, Uncategorized, tags: Peter Garland, Sarah Cahill
After the Wars
Peter Garland
Cold Blue Music
Sarah Cahill, piano
Cold Blue Music has released a new CD of piano music by Peter Garland titled After the Wars. Recorded by Sarah Cahill, After the Wars is one of a series of works she has commissioned that focus on the concept of peace. Each of the four pieces on this CD is based on a Chinese poem or Japanese Haiku and is built on sustained piano tones and chords. According to the composer the pieces in this album are "…simply stated with relatively little temporal or thematic development."
The first track is Spring View: "The nation is ruined, but the mountains and rivers remain." ( after Tu Fu) This begins with a series of deep, rumbling chords that paint a vivid picture of doom and destruction. These continue but are interspersed with higher, lighter chords that seem, in contrast, to hold out some hope. The heavy, sustained sounds boom out and then slowly fade after each passage. The lighter chords seem to be making cautionary comments on the devastation declared by the powerful sounds in the lower register. Spring View is the darkest of the pieces in this album and becomes the starting point for what follows.
The second track is titled "Summer grass / all that remains / of young warriors' dreams." (after Basho) and this also begins with dark, dramatic chords that ring out from the depths of the piano. In this piece, however, the chords climb up to the higher registers as if ascending a ladder. There is an ethereal feel to this progression, a redemptive quality that springs out of the previous darkness. The sustained ringing of the chords as they are struck hover in the air like spirits awaiting release. There is a more reflective feeling here and ultimately a sense of restful assurance.
Track three is Occasional Poem on an Autumn Day: "When I'm at peace I let everything go." (after Ch'eng Hao) and this has a pleasantly sunny feel after the heavy drama of the first two pieces. Dense, sustained chords open but turn warmer and more relaxed as the piece progresses. A sense of relief is felt and the touch of Sarah Cahill on the keyboard is precise enough to give a slightly different feel to each of what is a succession of very similar chords. At 3:45 the dynamics increase noticeably and the chords become joyful, like hearing the peal of bells. The tempo slows towards the close and the volume tapers down to a quiet, peaceful chord at the finish. Occasional Poem on an Autumn Day effectively expresses a welcome sense of placid release and calm.
The final piece on the album is "A snowy morning / smoke from the kitchen roof– / it is good."
(after Buson). This begins with high, bright chords and an appealing, sunny harmony that is followed by lush and comforting deep notes. There is a sense of peaceful happiness in the deliberate chords; the comforts of domestic tranquility fill up the sound. More deep chords, solemn but not sad, rise up to a sunlit landscape. All is now peaceful and harmonious.
The arc of After the Wars – from destruction and ruin to the return of placid domesticity – is artfully realized here with a minimum of musical materials and the exquisite playing of Sarah Cahill. Hearing this album creates in the listener a desire for that same healing process to be present in our own life and times. It inspires the hope that no matter how gloomy the present may seem – and our world is full of terrible things – the path to a peaceful wholeness is yet attainable.


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Pardale-V is an analgesic for use in dogs. It is suitable for use after injuries or in other painful conditions. It is traditionally used to help joint pain. It is not recommended to be given continuously; a 5 day course should be all that is needed, though repeat courses can be given after a suitable break. If in doubt, consult your veterinary surgeon. (NFA-VPS)
Below is the product datasheet.
Company name: Dechra Veterinary Products Limited
Address: (A business unit of Dechra Pharmaceuticals PLC)
Sansaw Business Park
Hadnall, Shrewsbury
Shropshire SY4 4AS
Telephone: 01939 211200
Fax: 01939 211201
Email: email@example.com
Website: www.dechra.com
White, flat tablets with a bevelled edge and a break-line.
Each tablet contains:
Active substances: Paracetamol 400 mg
Codeine phosphate hemihydrate 9 mg
For analgesic therapy in the dog only. The product is indicated for acute pain of traumatic origin, as a complementary treatment in pain associated with other conditions, and post operative analgesia.
For oral administration.
1 tablet per 12 kg body weight every 8 hours.
Small dogs (up to 6 kg body weight): Half a tablet every 8 hours.
Medium dogs (6-18 kg body weight): Half to one and a half tablets every 8 hours.
Large dogs (18-42 kg body weight): 1-3 tablets every 8 hours.
Treat for a maximum of 5 days.
Do not exceed the stated dose or the duration of treatment.
Do not administer other NSAIDs concurrently or within 24 hours of each other.
Use is contraindicated in animals suffering from cardiac, hepatic or renal disease, where there is the possibility of gastrointestinal ulceration or bleeding, or where there is evidence of blood dyscrasia or hypersensitivity to the product.
Do not use this preparation for cats.
Use in animals less than 6 weeks of age, or in aged animals, may involve additional risk. If such use cannot be avoided, animals may require a reduced dosage and careful clinical management.
Avoid use in dehydrated, hypovolaemic or hypotensive animals as there is a potential risk of increased renal toxicity.
Some NSAIDS may be highly bound to plasma proteins and compete with other highly bound drugs to produce an increase in non-bound pharmacologically active concentrations, which can lead to toxic effects.
Concurrent administration of potentially nephrotoxic drugs should be avoided.
There are no known contraindications for use during pregnancy.
If you notice any serious effects or other effects not mentioned in the data sheet, please inform your veterinary surgeon.
Overdose: Immediately seek the advice of a veterinary surgeon showing them the product literature.
Carry out lavage and treat with an intravenous injection of acetylcysteine (200 mg/ml) at a rate of 140 mg/kg every 6 hours for 7 treatments. Ascorbic acid (30 mg/kg) should also be given orally with each dose of acetylcysteine. If necessary, instigate fluid therapy using 'Ringers' or bicarbonate solution. Treat for codeine overdosing with intravenous injections of naloxone (1.0 mg/kg) repeated as necessary. Provide oxygen support.
Side effects: Occasional constipation due to codeine content.
Special precautions to be taken by the person administering the veterinary medicinal product to animals: Wash hands after use.
General precautions: For animal treatment only.
Keep out of the reach and sight of children.
Do not store above 25C.
Do not use after the expiry date stated on the blister.
Disposal: Dispose of empty packaging and any remaining product in the household refuse.
Containers of 100 and 500 tablets.
NSAIDs can cause inhibition of phagocytosis and hence in the treatment of inflammatory conditions associated with bacterial infections, appropriate concurrent antimicrobial therapy should be instigated.
Seek veterinary advice if the treated condition does not improve or worsens during treatment.
UK authorised veterinary medicinal product.
Dechra Limited, Dechra House, Jamage Industrial Estate, Talke Pits, Stoke-on-Trent, Staffordshire, ST7 1XW.
Vm 10434/4034.
All prices include VAT where applicable. *The freephone number is free from most UK landlines only, mobiles are usually charged so we'd recommend calling our landline from your mobile or internationally.
Below are some recent questions we've received regarding Pardale-V Tablets for Dogs, including answers from our team.Ask Your Own Question
Can this drug cause hyperactivity?
Hyperactivity is unlikely. It might through pain relief make a dog feel much more perky and happy. If your dog seems to show any sort of odd reaction to any medication, it is best to re-evaluate if the medication should be given any more. Your vet should be bale to help with this. If in doubt, stop the medication, then perhaps try it again after a week or so and see what result you get,
I have a fifteen year old collie cross who is on Pardale-v for Ten days but the vet has talked about leaving her on it with her Loxicom which she takes regularly . After reading the comments I Believe her body is to old to handle the drug on a permanent bases .
Pardale-V is a safe drug if used carefully and sensibly. Like many drugs it does have potential side effects but if your dog is on an appropriate dose and your vet has ascertained that there are no underlying conditions which affect it, the risks are very small. It also works well in combination with Loxicom and has the potential to greatly reduce the stiffness and discomfort your dog might otherwise feel. Dogs tend to suffer in silence but that does not mean they do not want to be made more comfortable. It is up to you and your vet, but I would be inclined to give it a try and see how your dog gets on. You should stop both medications if your dog starts to have any vomiting or diarrhoea or goes off her food, but otherwise I would give it, regardless of her age.
My husband accidentally took a quarter of one will it be harmful
Obviously this is really a question to direct at your own doctor. However the active ingredient is very safe to use in people (and at a higher dose than that) so I doubt if there are any concerns.
Can these tablets be given alongside to a dog who is on steroids for allergies?
These tablets are not recommended to be given with steroids. Although they are generally safe, that is a combination which might well cause problems, so I certainly would not do it.
My Labrador has been prescribed x3 pardale v daily for 2 weeks post hip operation. Is this ok as I noticed suggestions not use for more than 5 days? Should he use alternative pain relief?
This is a safe product for dogs, though there is potential for it to build up in the body over time, especially if accidentally over-dosed. The guidance you refer to is sensible for general situations. If your vet has prescribed this dose in full knowledge of your own dogs bodyweight and general health, then I would expect it will be safe and effective. There are other medicines which could have been used so I am not sure I would have suggested Pardale-V myself, but it does have good qualities. It is of course OK for you to question your own vet about it.
We have 11year old diabetic dog with heart problems and arthritis. She has been prescribed pardale-v 2 tabs x 3 daily. Is this excessive and what side effects is she likely encounter. She is on 21 units of insulin. Cardalis 15 mg daily, and Cardisure 5mg twice daily
Pardale-V tablets are very safe if given for just a few days. If they are used continuously for longer periods then side effects are possible and are usually vomiting/diarrhoea but can relate to liver or kidney damage. This is something you should talk to your own vet about. There are other treatments to help with arthritis which are much more modern and potentially better than Pardale V, though they are more expensive.
My Staffordshire bull terrier (Buster) has been prescribed half a tablet (pardale v) to treat a pain in his neck, either a muscular or disc problem. Looking at your website it suggests 1 tablet per 12kg bodyweight every 7 hours, Buster weighs 25kg so that suggest 2 tablets every 7 hours. Buster does suffer with acidity in his stomach are these tablets likely to make it worst?. Many thanks.
Your vet has prescribed a dose which is lower than that generally recommended by the manufacturers. I would presume that your vet has good reason to do this and so I would suggest you do not go against that without talking to your vet about it first. These tablets can be associated with stomach and other problems, and if given at the full dose, a maximum of a 5 day course is recommended at a time. The higher the dose and the longer they are given for, the greater the chances of some unwanted side-effect. They can be very helpful and effective though if used carefully.
Why can't it be given continuously
As with all effective medication there is a potential for side effects to occur. With this drug, liver damage is the main concern, though kidney and gastro-intestinal damage are possible. Problems are more likely to occur if a high dose is given every day. Damage can be a cumulative effect building up over time. The chances of this happening are reduced with a lower dose and a shorter period of treatment.The manufacturers only received a license for the dose and duration of treatment as recommended. The truth is that some dogs (and who knows which ones) are probably much more likely to suffer side effects than others. Possibly many dogs could safely take treatment for longer. However it is judged that a significant portion of dogs might be at risk from side effects for the 5 day limit to be in the license. More than that it is not possible to say, other than the fact that the product has been on sale for many years and is regarded as generally safe. I believe there are no great concerns about reports of adverse reactions or side effects.
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subset: commoncrawl


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Kaospilot Dead Conversation Lyrics
Last updated: 02/16/2004 09:06:25 PM
Another useless statement. This conversation is dead. We sit and talk for hours. But nothing is really said. You look at me: I look at you. "Everything is fine". So concerned about maintaing our pretty facade. But nothing is said. Pretty facade.
Click here to submit the Corrections of Dead Conversation Lyrics


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Perfect party game. Funny avatars. Simple menus. Custom controllers.
Level of trivia is a little low.
Bottom Line
Claiming your spot as the party trivia master just got more fun, thanks to Buzz Quiz TV pie-in-the-face-style punishments for incorrect answers.
Making a trivia game is easy. Rewriting the trivia formula, though, is a bit harder. While Buzz Quiz TV doesn't break new ground or hit on a whole new category of inane knowledge (its categories are, more or less, reworded slices of Trivial Pursuit pie), it does earn tons of points for presentation. The questions asked of the players by the animated Buzz (who is startlingly reminiscent of Whose Line Is It Anyway?'s Greg Proops, by the way) aren't really challenging, but the way you earn points (and take them from your competition) is nothing short of hysterical: One round is an animated game of hot potato with a lit bomb, another arms you with pies to knock out your competition, and a third lets the player who answers correctly swipe points from whomever they choose. The avatars include a cheerleader, disco dancer, a mime, and a Kill Billstyle vixen, and watching them interact is the biggest appeal of the game play itself. Aside from schooling your friends, of course.
For more on this game, see 1Up.com's review of Buzz Quiz TV.
More Computer & Console Gaming:
Deus Ex: Mankind Divided (for PC)
No Man's Sky (for PC)
Metal Gear Solid V: The Phantom Pain (for PC)
Skullgirls 2nd Encore (for PC)
Madden NFL 2017 (for PlayStation 4)


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- Read up on your hero
- Currently Unavailable
The former YTS trainee and boyhood Liverpool fan is one of the most successful products of the Liverpool FC youth academy. He has spent his entire career wowing the fans and gaining the respect of supporters and fellow professionals alike. England's number 4 stands ready to weave some of his magic on the pitch and be part of the driving force of England's World Cup team. 128 pages. By Adam Cottier.
Lamentamos que todavia no hay descripción en español para este producto.


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Job Overview
The mission of the U.S. Department of the Interior (DOI) is to protect America's natural resources for future generations, provide access to our nation's natural and cultural heritage, offer recreational opportunities, honor its trust responsibilities to American Indians and Alaska Natives, conduct scientific research, provide wise stewardship of energy and mineral resources, and conserve and protect fish and wildlife.
The Department of the Interior, Office of Inspector General (OIG) is an independent office within the Department of the Interior (DOI) whose mission is to promote excellence, integrity, and accountability throughout DOI. The OIG conducts investigations, evaluations, and audits in order to enhance effectiveness and efficiency and to prevent and detect waste, fraud, and mismanagement in DOI programs and operations.
As an Information Technology Specialist within OIG's Information Technology Division, Office of Management, you will support the unique mission of the OIG primarily in the area of information systems administration and development, oversight, telecommunications, and information management. You will play an important role in enabling the OIG to advance its mission of providing independent oversight and promoting excellence in the programs and management of DOI.
Selective Placement Factor: This position has a selective placement factor (SPF). SPFs are specific qualifications that are in addition to the minimum requirements for a position. Applicants who do not meet these requirements are ineligible for further consideration. Candidates must possess any current Certified Information Systems Security Professional (CISSP) certification from ICS2 to be considered. You are required to provide documentation to support your claim.
Duties: The selectee will act as a Network Administrator of the servers and applications unique to the OIG environment. In this capacity, the selectee will perform the following duties:
- Recommends installation of new or modified Network equipment (WAN or LAN) including communication lines, bridges, modems, multiplexes, network hardware and software for designing and implementing assurance measures to ultimately protect systems from intrusions or corruption by any inside/outside unauthorized entry.
- Using network management software, monitors network activity and tunes parameters where feasible to ensure optimum network performance. This includes monitoring all devices such as routers, hubs, and switches and data circuits.
- Monitors and evaluates existing telecommunications Cisco hardware and software for efficient and effective performance and recommends improvement measures as necessary. This includes in-depth analyses of all ongoing computer resource requirements, interfaces between existing systems and overall input on the network operating environment.
- Acts as a liaison to the Department regarding network integration and migration initiatives. This includes participating in conference calls and off-site meetings as necessary to stay informed of on-going developments and initiatives.
- Performs software maintenance and tuning to maintain optimum system performance and hardware utilization.


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Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis. Current treatment is rarely curative, thus novel meaningful therapies are urgently needed. Inhibition of Hedgehog (Hh) signaling at the cell membrane level in several cancers has shown anti-cancer activity in recent clinical studies. Evidence of Hh-independent Gli activation suggests Gli as a more potent therapeutic target. The current study is aimed to evaluate the potential of Gli as a therapeutic target to treat MPM. The expression profiles of Gli factors and other Hh signaling components were characterized in 46 MPM patient tissue samples by RT-PCR and immunohistochemistry. Cultured cell lines were employed to investigate the requirement of Gli activation in tumor cell growth by inhibiting Gli through siRNA or a novel small molecule Gli inhibitor (Gli-I). A xenograft model was used to evaluate Gli-I in vivo. In addition, a side by side comparison between Gli and Smoothened (Smo) inhibition was conducted in vitro using siRNA and small molecule inhibitors. Our study reported aberrant Gli1 and Gli2 activation in a large majority of tissues. Inhibition of Gli by siRNAs or Gli-I suppressed cell growth dramatically both in vitro and in vivo. Inhibition of Gli exhibited better cytotoxicity than that of Smo by siRNA and small molecule inhibitors vismodegib and cyclopamine. Combination of Gli-I and pemetrexed, as well as Gli-I and vismodegib demonstrated synergistic effects in suppression of MPM proliferation in vitro. In summary, Gli activation plays a critical role in MPM. Inhibition of Gli function holds strong potential to become a novel, clinically effective approach to treat MPM.
Citation: Li H, Lui N, Cheng T, Tseng H-HK, Yue D, Giroux-Leprieur E, et al. (2013) Gli as a Novel Therapeutic Target in Malignant Pleural Mesothelioma. PLoS ONE 8(3): e57346. doi:10.1371/journal.pone.0057346
Editor: Jingwu Xie, Indiana University School of Medicine, United States of America
Received: October 8, 2012; Accepted: January 21, 2013; Published: March 6, 2013
Copyright: © 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH/NCI grant R01CA125030; the Eileen D. Ludwig Endowed for Thoracic Oncology Research (to BH); NIH/NCI grant R01CA132566; the Bonnie J. Addario Lung Cancer Foundation; the Kazan, McClain, Abrams, Fernandez, Lyons, Greenwood, Harley & Oberman Foundation; Paul and Michelle Zygielbaum; the Honeywell Foundation; and the Jeffrey and Karen Peterson Family Foundation (to DMJ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Malignant pleura mesothelioma (MPM) is an uncommon but inexorably fatal cancer that arises from the surface serosal cells of the pleura and, less frequently, from the peritoneum [1]–[3]. Treatment of MPM with surgery, chemotherapy, or radiation therapy is rarely curative with a median survival ranging from 10 to17 months [2]. Despite some promising results, long-term survival with currently available treatment is rare [3], [4]. Therefore, novel meaningful therapies for MPM are urgently needed.
Currently, in spite of frequent observation of NF-kB, EGFR, and PI3K/AKT signaling deregulation in MPM cells, the molecular mechanism underlying tumorigenesis in MPM is poorly understood [1], [5]–[7]. The Hedgehog (Hh) signaling pathway has been implicated in a wide variety of cancers, including leukemia, lung, brain, skin, head and neck, liver, gastrointestinal, colorectal, pancreatic, prostate, mammary, ovarian and renal carcinomas[8]–[12]. Therefore, exploring the role of the Hh pathway in MPM and inhibiting its aberrant activation holds great promise to provide novel and effective treatments for MPM patients.
In the quiescent state of the Hh pathway, the twelve-pass trans-membrane receptor Patched-1 (Ptch1) restrains the activity of the seven-pass trans-membrane receptor Smoothened (Smo) [10], [12]. Binding of Hh ligands to Ptch1 reverses the inhibitory effect on Smo. Activated Smo elicits a complex series of cytoplasmic signal transduction events resulting in activation of the Glioma-associated oncogene (Gli) family of transcription factors. The Gli transcription factors then translate the extra-cellular Hh-stimulus into defined transcriptional programs in a context-dependent and cell-type specific manner [10], [12].
The aberrant activation of Hh signaling happens at several levels throughout the pathway, contributing to the development of many aggressive and metastatic cancers [12]. Conventionally, the frequent activation of the Hh pathway in tumors is thought to be mainly due to overexpression of ligands, loss of Ptch or constitutive active mutants of Smo [8], [10], [12]. Most efforts have been devoted to investigate the inhibition at the cell membrane level, i.e. Smo and Hh inhibitors [12]. The most clinically advanced example is vismodegib (also known as GDC-0449), which was newly approved by the U.S. Food and Drug Administration to treat adult patients with basal cell carcinoma [13]–[15]. Multiple clinical trials are evaluating the use of vismodegib in other types of cancer, as well as several other candidate drugs that target Hh signaling [12], [15].
Downstream Hh pathway activation has also been documented in tumors of the brain, prostate, muscle and in cell lines derived from pancreatic and lung cancers [9], [16]–[21]. The attributed molecular mechanism includes loss of other Hh pathway factors downstream of Hh/Smo and upstream of Gli, such as Sufu and Ren, and Gli gene amplification and chromosomal translocation. Furthermore, a growing body of evidence has revealed additional mechanisms of Gli activation which are independent of Hh/Smo regulation [22]. The Hh-independent Gli activation is stimulated by cross-talk between components downstream of Hh/Smo and several other oncogenic signaling pathways, such as the transforming growth factor β (TGFβ), epidermal growth factor receptor (EGFR), RAS and AKT/PI3K pathways [8], [23]–[32]. Overall, the concept that Gli proteins serve as an integration point of several signaling cascades, in addition to canonical activation from Hh/Smo, has significant implications for the understanding of tumor development. It strongly argues for the strategy to develop novel therapies that target Gli proteins in order to treat aggressive tumors, such as MPM.
The current study investigated the aberrant activation of Gli proteints in MPM, explored the effectiveness of targeted inhibition by a novel Gli inhibitor (Gli-I) to inhibit MPM cell growth, and compared the efficacy of Smo and Gli inhibitors. Our result strongly suggests that targeting Gli factors holds strong potential to become clinically effective treatment options for MPM patients in the near future.
Materials and Methods
Ethics Statement
The study with patient tissues was approved by the Committee on Human Research (CHR approval number: H8714-11647-10) at the University of California, San Francisco (UCSF). Written, informed consent was obtained from each patient before specimen collection. Mice study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Office of Ethics and Compliance of UCSF.
Patient Tissues
Tissue specimens were collected from 46 patients who underwent surgical resection for MPM at the Thoracic Oncology Program at UCSF. Samples were frozen immediately and stored in liquid nitrogen until use. Twenty-seven samples were fixed in formalin and embedded in paraffin to make tissue slides.
Immunohistochemistry, Immunofluorescence and Western Blot
Immunohistochemistry, immunofluorescence and western blot were performed following standard procedures. Antibodies applied to detect protein expressions were Gli1(Santa Cruz Biotechnology, Santa Cruz, CA), Gli2(Abcam, UK), SHh(Abcam), Smo (Sigma, St. Louis, MO), Ki67(Cell Signaling, Beverly, MA), active Caspase 3 (Cell Signaling) and Actin(Sigma). Total protein extraction was performed with M-PER Mammalian Protein Extraction Solution (Thermo Scientific, Waltham, MA), and 40 ug of proteins were analyzed in western blot.
RNA Extraction and RT-PCR
Total RNA was isolated from tissue or cultured cells using a RNeasy kit (Qiagen, Germany). Genomic DNA contamination was eliminated by DNase I treatment. Reverse transcription was conducted with 250 ng RNA using an iScript cDNA synthesis kit (Bio-Rad, Herculas, CA). The resulting cDNAs were analyzed with real-time RT-PCR using Gene Expression Assays in a 7900 Real-Time PCR System (Applied Biosystems, Foster City, CA) for 40 cycles (96°C for 15 seconds and 60°C for 1 minute). Gene expression was normalized to 18S expression. We defined the Ct value of negative controls (RT minus controls) as the baseline to calculate relative mRNA expression.
Cell Culture, Drug Treatment
Human mesothelioma cell lines NCI-H28, MS-1, REN, H2052, H2452 and H290 were purchased from the Cell Culture Core Facility at Harvard University (Boston, MA,USA). The cell lines were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells were seeded one day before treatment with Gli-I, cyclopamine (Selleck Chemicals) and vismodegib (Selleck) at different concentrations for 30, 48 or 72 hours, with vehicle (DMSO) as controls. Cells were subjected to the following analyses of immunofluorescence, RNA extraction and RT-PCR, TUNEL or proliferation assays.
Proliferation Assays and siRNA Transfection
Cells were treated with Gli-I, cyclopamine or vismodegib or transfected with siRNAs. Cell proliferation was monitored for 4 days with CellTiterGlo assay (Promega). Proliferation assays were performed for at least three times, and representative results were illustrated. Cells were transient transfected using Lipofectamine 2000 (Life Technology) with siRNAs at a total concentration of 50 nmol/L. In double siRNA treatments, the total siRNA concentration was the same as single siRNA treatments. All siRNAs were purchased from Life Technologies. The efficiency of siRNAs was evaluated by western blot.
TUNEL Assay
TUNEL assay was performed using the DeadEnd Flurometric TUNEL System (Promega) per standard protocol for both FFPE tissue sections and culture cells. REN, MS1 and H28 were treated with either Gli-I or vismodegib for 40 hr before TUNEL assays. Each experiment was performed for three times.
Mice Study
Nude mice were subcutaneously injected with 10 million MS-1 cells with BD Matrigel Matrix (BD Biosciences, San Jose, CA). Fourteen days after inoculation, mice were randomized to intraperitoneal injections of either Gli-I at 50 mg/kg or vehicle alone for 14 consecutive days followed by another 7-day observation. Tumor volumes were measured from day 10 to day 35 post-inoculation. Tumors and organs were dissected on day 35 to make paraffin blocks for follow-up analyses. All experiments were performed at the UCSF Preclinical Core.
Statistical Analysis and Combination Index Analysis
Two-sided student's t-test was performed for proliferation assays and mRNA expression analysis. A p value <0.05 was indicated as *, 0.01 as **, and 0.001 as *** in corresponding figures. The combinational effects were quantified using the Chou-Talalay Method to obtain the Combinational Index (CI), where CI <1, = 1, >1 represent synergism, additive effect, and antagonism respectively.
Gli Family of Transcriptional Factors are Expressed in Malignant Pleural Mesothelioma
We first investigated the gene expression of Gli family of transcriptional factors as well as key SHh pathway components in MPM patient tissues. Tissue specimens were collected from 46 patients who underwent surgical resection for MPM at the Thoracic Oncology Program at the University of California, San Francisco. Among all patients, 11 patients were female, and 35 were male, with an average (± standard deviation) age of 67.2±10.7 years. Histologic analysis showed 39 (85%) epithelioid, 2 (4%) sarcomatous, and 5 (11%) undetermined tumors. There were 5 (11%), 8 (17%), 11 (24%) and 3 (7%) tumors at stage I, II, III and IV respectively, with 19 (41%) undetermined. Tumor samples were collected from all 46 patients, and normal adjacent pleura samples were available from 7 patients. Formalin-fixed, paraffin-embedded tissues were available from 27 patients.
The protein expression of Gli1, Gli2, Smo and SHh were characterized by immunohistocehmistry (IHC), and scored on a scale of 0–3 (negative, mild, moderate and strong positive). Representative samples in each category of the four proteins were summarized in Figure 1 A. More than 90% of the samples were positive for Gli1, Gli2 and Smo, and a majority of them had moderate to strong expression (Fig. 1C). In contrast, only 47% of the samples exhibited mild SHh expression (Fig. 1B, C). The expression profiles were further characterized by quantitative RT-PCR (qPCR). The expression of 46 tumor samples was compared with that of 7 normal pleura for shh, smo, gli1, gli2, ptch1 and ptch2 (Fig. 2A–F). The average expression level of gli2 was 2.4 fold as high in tumor tissues as in patient normal pleura (p<0.01), whereas gli1 levels were statistically comparable (Fig. 2C, D). Consistently, Gli target genes ptch1 and ptch2 were 2.8 (p<0.01) and 3.4 (p<0.05) fold as high as in tumor tissues respectively (Fig. 2E, F). SHh, Smo, Gli1 and Gli2 were expressed in five tested MPM cell lines (Fig. 3). Strong expression of Gli factors and elevated expression of their target genes in tissues indicated that the aberrant Gli activation may play an active role in MPM.
, Representative protein expression of Gli1(first row), Gli2(second row) and Smo (third row). Immunohistochemistry staining was scored as 0–3. Representative images of score 1 (first column), 2 (second column), and 3 (third column) were shown. B, Representative SHh protein expression. Representative images of score 0 (left panel) and score 1(right panel) were shown. C, Expression profiles of Gli1, Gli2, Smo, SHh in MPM tissues. Percentage of score 0–3 of Gli1, Gli2, Smo and SHh was summarized.
, Statistic analysis of shh, smo, gli1, gli2, ptch1 and patch2 mRNA expression in normal pleura and tumor tissues. A total of 46 tumor tissues was compared with 7 patient normal pleura for its relative mRNA levels of shh, smo, gli1, gli2, ptch1, ptch2 by quantitative RT-PCR. Two-sided student's t-test was performed between normal and tumor tissues. gli2, ptch1 and ptch2 showed significant elevation (D–F). A p value <0.05 was indicated as *, <0.01 as **, and <0.001 as ***. The average expression of tumor tissues was labeled in the three genes with the average of normal pleura as 100% (D–F). G, Correlation analysis between shh and gli1 (upper) and gli2 (lower). The expression of shh was undetectable in 43% of samples, and thereby shown as the baseline level.
, Expression of shh (A), smo (B), gli1 (C) and gli2 (D) by quantitative RT-PCR. E, Protein expression of SHh, Smo, Gli1 and Gli2.
Interestingly, the expression of Gli factors was poorly correlated with their upstream stimulus SHh. Only 47% of the samples showed mild SHh expression, in contrast to more than 90% for Gli1 and Gli2 (Fig. 1C). At mRNA levels, shh was detectable in only 57% of the tumor samples, but also in 3 patient normal controls (Fig. 2A). When shh was plotted with gli1 and gli2 expression, no correlation was observed (Fig. 2G). No correlation between smo and gli1 or gli2 was observed either (Fig. S1). The lack of correlation was also confirmed in cultured MPM cells. No correlation was obvious between shh and its downstream factors gli1 and gli2 (Fig. 3A–D) at mRNA levels or at protein levels (Fig. 3E). Aberrant Gli1 activation and the lack of correlation with the upstream SHh signals in MPM prompted us to investigate the oncogenic role of Gli transcriptional factors.
Downregulation of Gli1 and Gli2 Inhibited Cell Proliferation in Malignant Pleural Mesothelioma Cell Lines
To investigate the tumorigenic function of Gli factors, we down-regulated Gli1 and Gli2 simultaneously to measure cell proliferation in MPM cell lines. We selected three cell lines to represent different expression profiles of key SHh signaling components: H28 had relatively high expression of Smo and SHh and low expression of Gli factors; whereas REN and MS-1 had the opposite expression profiles (Fig. 3). We suspected that H28 might harbor a dominant upstream SHh signaling, whereas REN and MS-1 might not. Suppression of Gli1 and Gli2 by two sets of independent validated siRNAs (Fig. 4E) resulted in significant inhibition of cell proliferation in 72 hours in all three cell lines (Fig. 4A–C), suggesting the tumorigenic role of Gli factors in MPM. Single treatment of gli1 or gli2 siRNA did not show significant inhibitory effects of cell proliferation (data not shown), which might be explained by the redundancy of the two transcriptional factors. The application of gli1 and gli2 siRNAs resulted in significantly better inhibition of cell survival than smo siRNAs in MS1 (p<0.05) and REN (p<0.001) (Fig. 4D), supporting Gli factors as potent therapeutic targets. Comparable effects were observed in H28 (Fig. 4D), which was consistent with the idea that active upstream SHh signaling presented in the cell line. Overall, the pro-proliferation function strongly suggested Gli factors as potential therapeutic targets.
, Cell proliferation was significantly reduced by double siRNA treatment targeting gli1 and gli2 in three MPM cell lines. Two sets of independent siRNA were applied in each assay. MTS assay was used to measure cell proliferation at 72 hours after siRNA transfection. D, Comparison of Smo and Gli inhibition at suppressing cell proliferation at 72 hours after siRNA transfection. Two-sided student's t-test was performed between control siRNA and gli siRNA (A-C), between gli siRNA and smo siRNA in REN and MS1 (D). A p value <0.05 was indicated as *, <0.01 as **, and <0.001 as ***. E, The efficiency of siRNA was monitored by western blot at 72 hours after transfection in REN MS1 H28. Each gene was knocked down with two independent siRNAs.
Targeting Gli1 and Gli2 by a Novel Gli Inhibitor (Gli-I) has Better Effects than Targeting Smo (vismodegib) in Reduction of Cell Viability
Our lab has developed a novel small molecule, the Gli inhibitor (Gli-I), which specifically inhibits Gli1 and Gli2 transcriptional activity, resulting in dramatic cytotoxicity in tumor cells that are Gli activity dependent [33]. We investigated the efficacy of Gli-I, and conducted a side-by-side comparison with two Smo inhibitors, vismodegib and cyclopamine, in MPM cell lines. Efficacy of Gli-I, vismodegib and cyclopamine were determined by cell proliferation assays in five MPM cell lines. The IC50 values of Gli-I ranged from 3.75 µM to 13.63 µM upon drug treatment for 72 hours, which were much lower than that of Smo inhibitors vismodegib and cyclopamine (Fig. 5A).
, IC50 comparison of Gli-I and the Smo inhibitors. Cells were treated with corresponding compounds at 7 different concentrations for 72 hours to obtain a dose-respond curve in order to determine IC50 values. B–D, Gli-I achieved a better suppression of cell proliferation than the Smo inhibitor in three MPM cell lines. MTS assay was used to measure cell proliferation at 72 hours upon drug treatment. Two-sided student's t-test was performed between DMSO and drug treated cells. A p value <0.05 was indicated as *, <0.01 as **, and <0.001 as ***. E, Apoptosis in Gli-I and vismodegib treated cells. TUNEL assays were performed at 40 hours after drug treatment, with TUNEL in green and DAPI in blue.
To confirm the cytotoxicity of Gli-I, we measured cell growth after Gli-I treatment. Cell proliferation was dramatically suppressed upon the treatment of Gli-I at concentrations lower than their corresponding IC50 values in different cell lines, i.e. 2.5 µM, 5 µM and 10 µM for REN, MS-1 and H28 respectively (Fig. 5B–D, p<0.001). In contrast, vismodegib showed no obvious effects when applied at the same concentration as Gli-I (data not shown), and had moderate effects in REN and MS-1 at 10 µM and 20 µM respectively, which were quadruple the concentration of Gli-I in the same assay (Fig. 5B, C). Vismodegib at 20 µM achieved comparable anti-proliferation effects as Gli-I at 10 µM in H28 (Fig. 5D). The comparison confirmed that H28 was sensitive to both Gli and Smo inhibitors, whereas REN and MS-1 was preferentially sensitive to Gli-I, which was consistent with the results of siRNA inhibition (Fig. 4). TUNEL assay was conducted in the cell lines upon 40 hr drug treatment at the same concentration as in the proliferation assay. Gli-I induced intensive apoptosis whereas vismodegib resulted in moderate apoptosis (Fig. 5E), which was consistent with the proliferation assay.
To verify the specificity of Gli-I, protein expression of Gli1 and Gli2 were evaluated by immunofluorescence (IF) upon drug treatment for 48 hours (Fig. 6A). For REN and MS1, the down-regulation of Gli1 and Gli2 was significant by Gli-I, and only moderate by vismodegib; whereas for H28, the down-regulation were at comparable levels by the two compounds. RNA levels were monitored upon 30 hr treatment, and the suppression of gli1 and gli2 by Gli-I was more effective than that by vismodegib in all three cell lines (Fig. 6B).
, Immunofluorescence staining of Gli1 and Gli2 upon 48 hr treatment of Gli-I and vismodegib in three MPM cell lines. B, RT-PCR analysis of gli1 and gli2 expression upon 30 hr treatment of Gli-I and vismodegib. The concentrations of both compounds for each cell line were the same in A and B, and were labeled in B.
Overall, the results strongly suggested that our novel compound Gli-I achieved a significant anti-proliferation effect by down-regulating Gli factors in vitro, and Gli factors might serve as more effective targets than Smo in treating MPM.
Gli Inhibitor Inhibits Tumor Growth in a Xenograft Model
To determine whether Gli inhibition represented a potent approach to suppress mesothelioma cell growth and tumorigenesis in vivo, the efficacy of Gli-I was examined in a MS-1 xenograft model. Fourteen days after implantation, when most tumors reached 100 mm3, mice started to receive daily IP injections of Gli-I at 50 mg/kg for 14 days. Gli-I treatment significantly inhibited tumor growth by 51% at the end of the 14-day treatment compared with the control group (Fig. 7A, p<0.05). In addition, the xenograft re-growth after treatment withdrawal was monitored for another 7 days. The inhibition of tumor growth was sustained and more substantial (63%) compared with control group (Fig. 7A, p<0.01), indicating durable effects of Gli inhibition. No obvious change of body weight was observed during the course, or any noticeable toxicity in major organs from the treated mice (data not shown).
, MS-1 xenograft tumor growth upon Gli-I treatment. Tumor size was measured every 3–4 days. Red arrow indicated the period of drug treatment. Tumor volume was calculated by using the equation x2y/2 (where x<y), and presented as percentage of its initial volume on day 14. Two-sided student's t-test was performed between control and drug treated mice at day 28 and day 35. A p value <0.05 was indicated as *and <0.01 as **. B, Gli1, Gli2 and Ki67 expression in the resected xenograft tumors by IHC. C, Apoptotic cells in the resected xenograft tumors by IF. Active Caspase 3 (red) with DAPI (blue) in two left columns, and TUNEL (green) with DAPI in two right columns.
In addition, we examined the resected xenograft tumor specimens after the completion of the in vivo experiment (Fig. 7B). Gli1 and Gli2 proteins were down-regulated in Gli-I treated tumors compared with control, consistent with in vitro results (Fig. 7B). The decrease of Ki-67 levels confirmed the reduction of proliferation in Gli-I treated tumors (Fig. 7B). Dramatically increased apoptosis was visualized by active caspase 3 staining and TUNEL assays (Fig. 7C). The xenograft study strongly suggested that Gli-I had an extensive anti-tumor efficacy in vivo by inhibiting Gli factors.
Gli-I has Synergistic Effects with Vismodegib and Pemetrexed Disodium
To further characterize the anti-tumor efficacy of Gli-I, we examined the combinational treatments of Gli-I plus vismodegib or the chemotherapy drug pemetrexed. We hypothesized that Gli-I might show a synergistic effect with vismodegib, as the latter may further suppress Gli activation in cells with active SHh/Smo signaling. In H28 where SHh-dependent Gli activation exists, the combinational treatment of Gli-I and vismodegib led to a significantly better cytotoxicity than either single treatment (Fig. 8A). The combinational effects were further quantified using the Chou-Talalay Method to obtain the Combinational Index (CI), where CI <1, = 1, >1 represent synergism, additive effect, and antagonism respectively. The CI analysis showed synergistic effects at lower dose, and additive effects at higher dose.
, Cells were treated with Gli-I, vismodegib and pemetrexed at the concentration indicated in the figure. Cell survival was measured by MTS assays at 72 hours of drug treatment. The combinational effects were further quantified using the Chou-Talalay Method to obtain the Combinational Index (CI), where CI <1, = 1, >1 represent synergism, additive effect, and antagonism respectively. Two-sided student's t-test was performed between single and double treatment. A p value <0.05 was indicated as *, 0.01 as **, and 0.001 as ***.
We also examined if Gli-I and pemetrexed might show synergistic effects. Gli activation has been suggested to maintain the stem cell population, which contributes to drug resistance and recurrence [34]. Therefore, it is possible that Gli-inhibition might improve the effectiveness of pemetrexed. Pemetrexed is one of the most commonly used first line chemotherapy drugs. Improvement of penetrexed efficacy would be beneficial to MPM patients. We observed a dramatic synergistic effect when Gli-I was applied together with pemetrexed (Fig. 8B).
Both studies suggested the combinational treatments as meaningful approaches to treat MPM, further supporting Gli factors as promising therapeutic targets in MPM.
Aberrant Gli activation has been implicated in tumorigenesis in a wide variety of tumors [22]. The current study reported the essential role of the Gli family of transcriptional factors for MPM tumor growth. First, a large majority of MPM tissues had significantly higher expression of Gli and their downstream transcriptional targets (Fig. 1). Secondly, significant suppression of MPM cell proliferation in vitro was achieved by inhibiting Gli by siRNAs (Fig. 4). Furthermore, the application of a novel Gli inhibitor (Gli-I), which specifically inhibited Gli factors, led to a dramatic reduction of MPM cell proliferation both in vitro and in vivo (Fig. 5, 6, 7). All the evidence highlighted Gli as a potent therapeutic target in MPM.
Our results showed that directly targeting Gli1 and Gli2 achieved greater cytotoxicity than targeting Smo in MPM cells. Inhibition of Gli by siRNAs was more effective than that of Smo to suppress cell proliferation (Fig. 4). In addition, Gli-I was more potent to reduce cell growth than two Smo inhibitors, vismodegib and cyclopamine (Fig. 5). Furthermore, Gli-I reduced Gli levels more effectively than vismodegib (Fig. 6). In fact, the reduction of Gli levels by vismodegib was moderate even at a much higher concentration (2–4 fold higher than Gli-I).
What are the possible explanations for the difference in effectiveness of Smo and Gli inhibition? It is possible that inhibition of an upstream factor such as Smo might not effectively translate into the reduction of downstream Gli factors. However, we favored the explanation that the better efficacy was likely due to the Hh-independent Gli activation in MPM. In other words, Smo inhibition had little effect on Hh-independent Gli activation, and thereby was less potent in growth inhibition of cancer cells lacking upstream canonical Hh/Smo signaling. The coexistence of Hh-dependent and-independent Gli activation has been documented by a growing body of evidence in a variety of cancers, and the underlying mechanisms of Hh-independent Gli activation involved multiple signaling pathways, such as TGFβ, EGFR, and RAS and AKT/PI3K pathways [9], [22]. It is likely that MPM employs both Hh-dependent and -independent Gli activation in a context dependent manner. Our findings of elevated Smo and SHh expression in some tissues, as well as suppression of cell proliferation through the inhibition of Smo via siRNA and small molecule inhibitors, suggested that the conventional Hh pathway plays a role in MPM. However, the lack of correlation between SHh and Gli expression in MPM patient tissues and cultured cell lines indicated that the upstream signals from SHh through Smo might not be the only stimuli of aberrant Gli activation. Consistently, we observed differentiated responses to Smo and Gli inhibition in the three MPM cell lines. It is possible that H28, which had a comparable response to Smo and Gli inhibition, relied on the Hh-dependent Gli activation, whereas REN and MS1, which preferentially responded to Gli inhibition, harbored the Hh-independent Gli activation to a significant extent. The molecular mechanism underlying potential non-canonical Gli activation in MPM were currently under investigation in our group, and might further support Gli as a potent therapeutic target to treat MPM.
A very recent publication by Shi et.al documented the essential role of Hh signaling in MPM, and suggested Smo inhibition as a therapeutic approach [35]. Our study provided a different perspective to understand the molecular mechanism of MPM, and suggested Gli inhibition as a promising approach to treat MPM patients. We reported different expression profiles of the Hh pathway components, for example, SHh, Gli1 and Gli2. The difference is possibly due to the small patient numbers, and the limited normal pleura samples that were used as the expression baseline. However, we suspected that the existence of the Hh-independent Gli activation might complicate the overall expression profiles and thereby contribute to the different expression profiles reported by the two studies. Moreover, Shi et.al documented MPM cell lines that were insensitive to Smo inhibition. It would be interesting to examine if Hh-independent Gli activation plays a role in these cell lines and if these cell lines respond to Gli inhibition.
Moreover, both in vivo and in vitro studies strongly suggested that the novel Gli inhibitor, Gli-I, held a great potential to be a potent therapeutic agent to suppress tumor growth. Our group has accumulated a large body of evidence suggesting that the small molecule specifically and effectively inhibited Gli1 and Gli2 both in vivo and in vitro, dramatically suppressed tumor growth in several tumor types, such as lung cancer, melanoma and MPM as reported here, and had a very low toxicity profile in vivo. The synergistic effects of Gli-I with other therapeutic compounds also indicated the potential benefits to further develop the novel Gli inhibitor.
In summary, the Gli family of transcriptional factors plays a critical role in MPM. Inhibition of Gli is highly effective at suppressing cell proliferation in MPM cells in contrast to targeting the upstream factor Smo. Our small molecule Gli inhibitor, Gli-I, has a strong potential to become a novel, clinically effective approach to treat MPM.
Supporting Information
Figure S1.
Correlation between Smoothened and Gli. Expression of smo and gli1 gli2 was quantified by qPCR. The expression of smo was plotted against gli1 (upper) and gli2 (lower).
We would like to thank Dr. Byron Hann, Dr. Don Hom, Dr. Donghui Wang and Dr. Paul Phojanakong at the UCSF preclinical core for their contribution to the xenograft experiments. We also thank Dr. Il-Jim Kim and Helen Choi for their generous help with patient tissue process.
Author Contributions
Conceived and designed the experiments: HL NL BH DMJ. Performed the experiments: HL NL HTD DY TC HKT EGL QS JJ TL DY. Analyzed the data: HL NL BH DMJ. Wrote the paper: HL BH DMJ.
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