Archive for the ‘Dog Osteosarcoma’ Category

Toxic levels of chemical found in dog foods

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Posted 09 Jan 2012 — by James Street
Category Dog Osteosarcoma

Toxic amounts of a fluoride have been found in several major brands of dog food, possibly putting pets at a higher risk of cancer, neurotoxicity and other life-threatening illnesses, a research organization warns.

The dog foods contain fluoride levels 2.5 times higher than the Environmental Protection Agency’s national drinking water standard and those excessive levels “can predispose dogs to health problems, along with high veterinary bills, later in life,” according to the Environmental Working Group.

“Due to a failed regulatory system and suspect practices by some in the pet food industry, countless dogs may be ingesting excessive fluoride that could put them at risk,” Olga Naidenko, lead researcher of the Environmental Working Group-sponsored study, states in a media release.

Scientists have yet to determine how much fluoride is safe for dogs, but they have found people who consume excessive fluoride often develop mottled teeth (dental fluorosis) and weakened bones, leading to more fractures. High fluoride consumption is also associated with reproductive and developmental system damage, neurotoxicity, hormonal disruption, and bone cancer.

Three studies show that boys ages 6 to 8 who drink fluoridated tap water face a heightened risk of osteosarcoma, the rare but deadly form of bone cancer associated with fluoride. Scientists suspect that boys’ rapid growth may make them more susceptible to bone cancer.

Dogs may be even more vulnerable to osteosarcoma than humans, according to EWG. More than 8,000 osteosarcoma cases occur in dogs each year in the United States, nearly 10 times the number that occur in people, according to the study.

“Whatever the size and the appetite of a dog, combined fluoride exposure from food and water can easily range into unsafe territory,” the study states. “And, unlike children, who enjoy a variety of foods as they grow up, puppies and adult dogs eat the same food from the same bag every day, constantly consuming more fluoride than is healthy for normal growth.”

In the study, 10 brands of dog food were tested. Two dog food brands, one with vegetarian ingredients and one made by a small manufacturer, had no detectable levels of fluoride. But eight others – all major brands – found to contain high levels of fluoride. The contents of those brands included chicken byproduct meal, poultry byproduct meal, chicken meal, beef and bone meal. Any ingredient described as “animal meal” is basically ground bones, cooked with steam, dried, and mashed to make a cheap dog food filler.

The Washington-based Environmental Working Group, whose stated purpose is to protect human health and the environment, advises pet parents to feed food to their dogs that contains no bone meal and other meat byproducts to minimize exposure to harmful pollutants, including fluoride. “To protect pets from excessive fluoride exposures, dog owners can purchase pet foods that do not contain bone meal and other animal byproducts,” the study states.

Pet food should be held to the same health and safety standards as human food and should be free of contaminants that may endanger pets’ health, the study states. Yet, the federal Food and Drug Administration has little authority and few resources to ensure that products produced for pets are safe. The fact so many popular national pet food brands contain previously undetected health hazards shows that better federal food safety regulations are needed.

Vitamin D could help in fighting pediatric bone cancer

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Posted 17 Dec 2011 — by James Street
Category Dog Osteosarcoma, Local Recurrence, Lung Metastases, Metastases, Osteosarcoma, Vitamin D

Posted by Laura HerringDecember 16, 2011 at 9:05 a.m.

A study by a group of Kansas University researchers found that vitamin D can cause cancerous bone cells to turn to normal bone cells.

The findings, which were published in the Journal of Orthopaedic Research, could lead to a new treatment in fighting pediatric bone cancer, which has a survival rate of 60 percent to 70 percent.

Recent studies have shown vitamin D can inhibit the growth of malignant cells in breast, prostate and colon cancer. Kim Templeton, an orthopedic surgeon at Kansas University Hospital, was among the experts on a panel that discussed vitamin D research and cancer. She was surprised that none of the studies or trials included the effect of vitamin D on osteosarcoma, a malignant bone tumor that mainly affects children and adolescents.

“It’s the most common type of bone cancer in kids and teenagers and vitamin D is critical to bone health,” she said. So an interdisciplinary team at the Kansas University Medical Center came together to study how vitamin D affects bone cancer. The team used cancerous tumor cells to do the research.

“My question was if the tumor recognizes Vitamin D and if it would help control the cells,” Templeton said. In the laboratory tests, not only did the cancerous cells recognize the vitamin D, but it prevented the osteosarcoma cells from replicating as quickly and promoted the growth of normal bone cells.

“What should happen and what does happen (in the lab) is always two different things,” Templeton said. “So, I was happy it turned out the way we thought it would.”

The findings are important for a cancer who hasn’t seen the treatment methods or rate of survival change in the past 20 to 25 years. Most osteosarcoma patients undergo 10 weeks of chemotherapy before the tumor is removed.

The findings suggest that a normal size dose of vitamin D could become another tool in the treatment of osteosarcoma. Unlike chemotherapy, normal doses of vitamin D don’t have any negative side effects and it is inexpensive.

Before clinical trials on humans can began, researchers would have to test the effects of vitamin D on animals, which might include large dogs since they have a high rate of osteosarcoma.

Templeton said the findings don’t suggest people should start taking vitamin D to prevent bone cancer. Although that is a connection researchers might study in the future.

By Christine Metz

New studies help extend the lives of dogs with cancer

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Posted 05 Dec 2011 — by James Street
Category Dog Osteosarcoma
The College of Veterinary Medicine will begin a new trial this week.
Luke enjoys playing with a tennis ball while his owner,Track Huston, sits beside him Saturday at home in Crystal, Minn. Luke, a 6-year-old English Springer Spaniel, was the first participant in a trial for dogs with lymphoma at the University of Minnesota’s College of Veterinary Medicine.
Anthony Kwan
Published: 2011-12-05
Rachel Raveling rraveling@mndaily.com

When Luke, the Huston family’s 6-year-old English Springer Spaniel, was diagnosed with lymphoma, their vet recommended a new trial at the University of Minnesota’s College of Veterinary Medicine.

Luke was the first participant in a trial for dogs with lymphoma, but he’s one of many animal-companion owners bring to the University to contribute to research.

This week, the College of Veterinary Medicine will begin a new trial aimed at the latest treatment for dogs with osteosarcoma, a cancerous bone tumor.

Osteosarcoma often occurs in dogs’ front or hind legs, causing pain and bone destruction. It has high potential to spread to other parts of the body, said Antonella Borgatti, assistant professor of oncology and a researcher for the trial.

Catherine St. Hill, assistant professor of veterinary clinical science, said there are carbohydrates attached to cancer cells that make it easier for them to bind tightly to blood vessels — their mode of transportation through the body.

The goal of her research is to discover a way to either prevent the making of the carbohydrate or slow the progression of the disease.

When dogs pass away from osteosarcoma, it is most commonly a result of spreading, Borgatti said.

The OSAL —  osteosarcoma and salmonella —trial aims to stop the spread of cancer through amputation of the infected limb followed by chemotherapy and treatment using genetically modified salmonella designed to attack only the cancerous cells.

Kathy Stuebner, research coordinator for the University’s Clinical Investigation Center, said researchers will use PET-CT, a full body scan used to detect cancer and cancer spread.  It will be used on dogs for the first time before and after the treatment to help detect its effectiveness and identify proper treatment levels.

The goal for all their trials is to keep the dogs comfortable and prolong remission, she said.

Stuebner said the trial is “approved and ready to go.”  They hope to start this week.

In the meantime, five other oncology trials are active.  The trial that Luke initiated, called “Licking Lymphoma,” is testing Valspodar, a study drug, which Borgatti said should decrease the resistance to chemotherapy.

The trial was designed by Jaime Modiano, professor of veterinary clinical science, in partnership with Purdue University and the University of Pennsylvania.  Modiano said the trial was designed out of “scientific curiosity” and it “is transformational because it combines companion animals and human benefit.”

Dogs with lymphoma —cancer of the lymph nodes — go through “staging,” or full body testing, with X-rays and biopsies to determine the stage of their cancer.  Generally, healthy dogs with “B cell” lymphoma are accepted for the study, Borgatti said.

The dogs are put into two groups, some are given Valspodar and others are given a placebo, for four days.  Then they all go through chemotherapy.

The Huston family got involved in the “Licking Lymphoma” trial in April when they noticed their dog Luke had swollen lymph nodes in his neck.

Mike Huston, Luke’s owner, said they took him to the vet thinking he was suffering from allergies.  After a blood test, they were told that Luke might have lymphoma.  Their vet said Luke might meet the criteria to be a participant in the University’s lymphoma trials.

Huston said they took Luke to the University to be tested and after a long discussion as a family they decided to apply for the trial.

After the treatment Huston said Luke went into remission for seven months.

Borgatti said that most dogs only go into remission for a few months, so in Luke’s case, the trial was a success.

“Luke never showed any signs of pain, even during treatment.  He still runs and plays ball, everything is the same,” Huston said.

He added that the experience was much easier than he imagined because the veterinarians were very professional, yet personable.

“If he was going to get treatment, we decided we should do it there because the facility is the best and he could contribute to future research,” he said.

All studies that are done in the CVM are approved by the Institutional Animal Care and Use Committee, who check for ethics and animal welfare, which comforts many pet owners, Stuebner said.

There is also a financial incentive for owners to help pay for the chemotherapy in the oncology trials.  The “Licking Lymphoma” trial credits the owner $2,500 after he or she makes an initial payment before treatment.

“It’s a hard decision to make and most people wouldn’t pay for treatment for their pet without financial incentive,” Borgatti said.

There are free social services available to all clients who need extra support and help making a decision about what is best for them and their pet, she said.

She added that “most owners are happy that the trial is giving them an option.” By participating in research, they get more personal attention than they would at a normal vet.

“The trials give us a chance to get to know the owners really well,” Stuebner said.

Stuebner and Borgatti said the lymphoma trial has been going on for about six months and based on their expectations, they said they hope the study will be finished in six months to a year.

The trials being done for dogs in the CVM are unique because they hope the research will be used for humans with similar diseases in the future, Borgatti said.

“It is very satisfying because if we’re wrong, we learn from it,” Modiano said. “And if we’re right, we’ve got something that works better than anything else.”

Luke’s lymphoma has returned, but like always, he still enjoys playing around with his family. The Hustons are working with the University to keep him comfortable as the disease progresses.

Dogs With Cancer Helping to Find a Cure

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Posted 03 Dec 2011 — by James Street
Category Dog Osteosarcoma, Osteosarcoma, Vaccine, vaccine

Dogs receiving various treatments are helping medicine find new therapies for people, too

December 2, 2011

When her black Lab, Emmy, started limping in 2008, Kathi Streeter suspected the normal aches and pains of aging. Then came the devastating diagnosis: osteosarcoma, a deadly bone tumor. Osteosarcoma affects humans, too—mostly children, whose long-term survival rate, if the cancer spreads, is under 40 percent. Though Emmy died in May at the ripe old age of 13, she gained nearly three years of healthy living, and one day her treatment may help those kids.

[Learn more about how dogs help mankind in Mysteries of Science: Amazing Animals.]

In her quest to save Emmy, Streeter learned about a study underway at Colorado State University’s Animal Cancer Center in Fort Collins, about 100 miles from her home in Franktown, Colo. It was testing a gene therapy that could be injected straight into osteosarcoma tumors. The gene delivers a molecule designed to induce the cancer cells to self-destruct. Veterinarians there wanted to see how well dogs reacted to the treatment, as part of an effort to determine whether it might also be investigated for use in children.

Streeter is a cancer survivor herself—in 2004, she underwent a double mastectomy and chemotherapy to treat breast cancer—and didn’t hesitate to sign Emmy up. After the injection, CSU vets gave Emmy the standard treatment, too: amputation of her leg plus six rounds of chemo. They’re now evaluating how the injection affected the tumor. Although the results of this trial have not yet been published, previous trials suggest that the therapy may enhance the immune system’s ability to combat the tumor.

CSU is one of 20 participants in the Comparative Oncology Trials Consortium (COTC), a growing program started in 2003 and managed by the National Cancer Institute to study cancer in dogs and to recruit them for clinical trials of new treatments. The goal is more effective, more personalized treatments for man as well as his best friend. “Several tumor types in dogs mimic human cancers in their biologic behavior and genetic signature,” says Susan Lana, associate professor of clinical oncology at CSU. “Dogs can help us try to answer questions like, ‘Why does this cancer spread?’ and ‘Are there genetic pathways we can explore for treatment?’ ”

Dogs are ideal models, Lana says, because they’re genetically similar to humans and share the same environment. They develop cancer naturally, unlike mice and rats, which must be engineered to have the disease. And dogs are big enough to undergo MRIs as well as blood tests and biopsies, so scientists can better observe changes in the cancer over time. Thanks to advances in genomics and gene sequencing, researchers have established which canine cancers are most similar to their human counterparts. Besides osteosarcoma, they include prostate and breast cancer, melanoma, soft tissue sarcoma, and non-Hodgkin’s lymphoma.

Vaccine success. Comparative oncology has already produced some success stories. In 2010, the U.S. Department of Agriculture approved Oncept, a therapeutic vaccine for dogs with melanoma. Therapeutic vaccines are designed to mobilize the immune system to make antibodies against cancer cells, which ideally then destroy the cells and keep the cancer from coming back, and they’ve long been the holy grail of cancer drug development. But many of the vaccines tested have proved disappointing. If Oncept is any indication, dogs might hold the key to fine-tuning cancer vaccines. Some dogs in the Oncept trials lived more than a year after their diagnosis—far outpacing the typical lifespan of one to five months with conventional therapies.

The data from the dog trials were impressive enough to prompt the Food and Drug Administration to green-light a small human trial of a similar drug. Jedd Wolchok, a physician at Memorial Sloan-Kettering Cancer Center in New York and the drug’s codeveloper, is hoping a pharmaceutical company will fund the large clinical trials that would be needed to get the human version of the vaccine approved. “These trials can take over five years and they’re exorbitantly expensive, but the risk could lead to a long-term payoff,” he says.

Veterinarian Gerald Post learned the benefits of canine cancer trials as a pet owner. “Instead of living three months, he lived 2½ years,” Post says of his miniature schnauzer, Smokey, a participant in the Oncept trials. “He taught me to leave no stone unturned.” Post is now an investigator for several canine clinical trials, which he runs out of his Norwalk, Conn., office.

Joining a trial offers twin rewards for dog owners: access to cutting-edge treatments they might not otherwise be able to find or afford, and, even when there’s little hope, the satisfaction of contributing to the quest for cures. “We knew the trial wouldn’t resolve the cancer,” says Richard Liscinsky, whose golden retriever, Samantha, 6, was part of a one-week trial of a protein-based lymphoma drug designed to restrict the growth of cancer cells. Liscinsky and his wife, Ann, who live in Bronxville, N.Y., hoped the treatment regimen would offer up some answers and give them one more summer in Vermont with their beloved pet. Lymphoma is all too common in golden retrievers; Samantha is the second of the three goldens the Liscinskys have owned that has contracted the disease. “It’s frightening that cancer is so rampant—for all of us,” Ann says.

Owners who participate in trials typically get at least part of the care at no charge. The funding comes from a variety of sources, including federal grants, pharmaceutical companies, and philanthropists with a soft spot for dogs. Among the last group are Dave and LuAnn Runkle of Wayzata, Minn., who lost their golden retriever, William, to a rare and aggressive form of cancer called histiocytosis and then launched the Will-Power Cancer Research Fund to support comparative oncology trials at the University of Minnesota. The $10,000 they’ve raised so far is helping to fund trials in both dogs and cats, which also develop tumors that are similar to human cancers. Dave’s motto? “Help your animal, help yourself,” he says.

Shelter rescues. Some comparative oncology programs are reaching out to dogs that have no owners to rely on. In the summer of 2009, the University of Pennsylvania’s School of Veterinary Medicine launched the Shelter Canine Mammary Tumor Program, for example. Veterinarians there rescue dogs with mammary tumors from shelters, remove the tumors, and then adopt the dogs out to local families. More than 30 dogs have benefited so far, says Karin Sorenmo, Penn Vet’s chief of medical oncology.

Sorenmo’s team is studying the tumors to try to figure out what causes benign breast cells to turn malignant and spread. “It’s metastasis that kills the cancer patient,” Sorenmo says. “If we can learn what genetic events make tumors spread, it opens up a lot of possibilities for new treatments.”

For Mildred Edmond, that possibility is intriguing on several levels. Edmond adopted Cali, a 6-year-old bichon frise in the Penn trial who had 11 tumors removed. “Poor little Cali—she had a full mastectomy,” Edmond says. Edmond herself survived breast cancer six years ago, so she is eager for the scientists at Penn to unravel the complexities of the disease. “I have two granddaughters and a great granddaughter. I’d hate for them to go through what I went through,” she says. (Edmond and Cali are both now cancer-free.)

Dog survivors sometimes play more than a research role. After Emmy survived her bout with cancer, Streeter signed her up for a program at Children’s Hospital of Denver called YAPS, for Youth and Pet Survivors. With Streeter’s help, Emmy sent letters and photos to a young girl being treated for brain cancer. “I became [the patient's] pen pal,” Streeter says. “She brought pictures of Emmy to surgery.”

Streeter likes to think that giving Emmy the opportunity to contribute to a fuller understanding of a cancer that affects kids made the disease more bearable for everyone involved. Emmy loved children, she says. “If I could have asked her permission to do the trial, she would have said, ‘Yeah, let’s do it.’ ”

Arlene Weintraub is the New York editor of Xconomy.com.

This story is excerpted from Amazing Animals, a U.S. News & World Report special edition. You can order it at www.usnews.com/animalsbook or by calling 1-800-836-6397.

Dogs Fighting Cancer

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Posted 02 Dec 2011 — by James Street
Category Dog Osteosarcoma, Lymphoma, Vaccine

Reported December 2011

FORT COLLINS, Colo. (Ivanhoe Newswire) –In the U.S, one in four people will die of cancer each year, but it’s not only humans that are in danger. Cancer is the leading cause of death among older cats and dogs. Now, new research is helping man’s best friends thrive while giving researchers a chance at curing cancer in humans.

Millie Edmonds always wanted to adopt. So when the opportunity knocked, she took it.

“She just needed someone to love her,” Millie Edmonds told Ivanhoe.

And that love was put to the test soon after Millie took Cali home. Cali had twelve tumors in her mammary glands

“We were there to help her – whether she was sick or not,” Edmonds said.

Like breast cancer in humans –early detection can save a dog’s life. That’s why oncologist, Dr. Karin Sorenmo created the Shelter Canine Mammary Tumor Program. She and her veterinary students provide care to shelter dogs with tumors. They collect the canine tissue samples for scientists to compare with human ones. Most dogs have tumors in one gland and will develop others. Researchers can study tumors in all stages of development…potentially stopping the spread of the cancer cells.

“If we can figure out what happens when a tumor becomes malignant, what are the most important genetic alterations, maybe there will be a target that can be drugged,” Karin Sorenmo, an oncologist at the University of Pennsylvania School of Veterinary Medicine told Ivanhoe.

At a clinical trial at Colorado State University Animal Cancer Center, oncologist Jenna Burton is helping dogs fight B cell lymphoma.

“Lymphoma is a very aggressive type of cancer and most patients are no longer with us 4 to 6 weeks of diagnosis,” Jenna Burton a veterinary oncologist told Ivanhoe.

In the trial, doctors combine two different types of chemotherapy drugs with a vaccine made from the patient’s own tumor.

“Using a patient’s own tumor to create a vaccine against it is something of interest to both vet and human oncologists,” Burton said.

Lab tests showed that when the vaccine was mixed with the drug Clodronate, it significantly enhanced tumor responses. The top three cancers in dogs are mast cell tumor, lymphoma and osteosarcoma–two of which also affect humans.

“Dogs are really good test subjects, a lot of people may not realize that dogs develop cancer just like people do,” Burton said.

She’s hoping looking at old drugs in a new way in animals, can give us a peak into the future of cancer treatment.

“There’s a lot of interest in ways we can manipulate the immune system in patients and dogs with cancer,” Burton concluded.

Saving our furry friends so they can save us.

Dogs that are not spayed are at least four times more likely to get mammary tumors. Lymphoma can affect any dog of any breed at any age. It accounts for 10 to 20 percent of all cancers in dogs.

Click here to Go Inside This Science and View Video or contact:

Sandy Van
Media Relations
sandy@prpacific.com

Inventors find way to insert chemo drugs into bones, offering new chances for cancer treatment

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Posted 26 Nov 2011 — by James Street
Category Drug delivery to bone, Osteosarcoma

BY BRANDON JOHANSSON Staff writer | Posted: Friday, November 25, 2011 11:16 am

AURORA | From its small office at the Fitzsimons Life Science District, MBC Pharma is imagining big things in the field of cancer treatment.

The startup, led by doctors Alexander Karpeisky and Shawn Zinnen, is developing a patented technology aimed at delivering cancer treatment drugs directly to the bone — a practice that has shown promise in recent animal trials.

The two doctors, who have each spent decades in research and the pharmaceutical business, said MBC’s technology uses bone-homing drugs called bisphosphonates that are chemically linked to cancer treatment drugs, to deliver cancer-fighting medicine directly to the bone, which can act as a broadcaster for the rest of the body.

Karpeisky said the company’s technology fills an important need because conventional chemotherapy cannot be concentrated on the bone in amounts that are pharmacologically relevant.

“The idea behind the compounds are actually very simple, elegant, and by that, genius, of course,” Karpeisky said with a laugh.

The company is based in the Fitzsimons Life Science District, just across East Montview Boulevard from the Anschutz Medical Campus. Zinnen said the location is ideal for the startup because it means quick access to research equipment on the campus, equipment that a young company could never hope to buy on its own.

MBC’s technology is targeted primarily at bone diseases, and Zinnen said recent research is proving that the bones are a crucial element in other diseases, too.

“The bone and the bone marrow in particular has a huge role throughout cancer development,” he said. “It can act as a sanctuary for tumor cells to reside before they go to metastasize other tissues.”

That means MBC’s technology could prove beneficial to treating several forms of cancer and other diseases, Zinnen said.

“People have started to say in the last couple years, bring drugs to the bone, that’s critical. Not just for killing cancer cells at the site, but it could be critical for the whole disease  path,” he said.

The company, which is still seeking investors, plans to expand its research into treating other diseases.

“We are possibly expanding. With investment, we know we will expand,” Zinnen said.

The company hopes to file an application with the FDA seeking permission to test the technology in humans within six months, Zinnen said. If everything goes as planned and the company gets the investments it needs, Zinnen said the product could be tested on humans within a year.

Like many biotech startups, MBC is trying to launch its technology in an environment that doesn’t have the venture capital opportunities it once did.

“If we were at this stage 10 years ago, we wouldn’t have trouble getting funds,” Zinnen said.

Zinnen and Karpeisky said the company is particularly excited about the future because of the success of recent animal trials.

Zinnen said MBC has tested its treatment on dogs with spontaneous osteosarcoma, and MBC’s treatment’s showed dramatic reduction in the size of tumors.

While the company can’t yet test the products in humans, Karpeisky said seeing MBC’s technology work so well in an animal was exciting.

“The fact that we see the very pronounced and significant biological activity in a natural disease in dogs is very, very encouraging,” he said.

But it’s not just that delivering drugs directly to the bone has clear treatment benefits, Zinnen said. The process also provides some relief for patients as they live with the grueling pain associated with tumors.

“Very quickly on our drugs, we had all the dogs tested have their pain go away,” Zinnen said. “For the owner, certainly for the dog, that’s a big deal.”

For more on the company, visit www.MBCpharma.com

One lucky dog: Cancer treatment saves pooch’s leg

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Posted 21 Nov 2011 — by James Street
Category Dog Osteosarcoma, radiation

By VIMAL PATEL
vimal.patel@theeagle.com

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S.Villanueva
Veterinary technician Autumn Brown prepares Rowdy, a dog suffering from a cancerous tumor in his leg, for surgery Friday at the Texas Institute for Preclinical Studies on the Texas A&M campus.

 

An 8-year-old Great Pyrenees named Rowdy was the recipient Friday of a cutting-edge surgery at Texas A&M to treat bone cancer.

Veterinarians used a specially designed microdrill to poke into the 81-pound dog’s front right leg to inject radioactive cancer-fighting isotopes directly into the tumor.

The osteosarcoma tumor generally has a 90 percent fatality rate, and the current standard of care is to amputate the leg and follow with chemotherapy.

But removing a leg from a pooch Rowdy’s size can be limiting and painful, and long-term success isn’t guaranteed, vets said at the Texas Institute for Preclinical Studies on Raymond Stotzer Parkway.

So owners were reluctant to amputate, instead searching for relief and control, if not a cure, of the tumor.

“We’re just trying to give these dogs a better quality of life for as long as we can,” said Theresa Fossum, director of TIPS.

The procedure is known as liquid brachytherapy. The surgeon used a tiny drill with a diameter of .017 of an inch that was designed by Houston-based Valco Instruments

Rowdy, who was unconscious during the surgery with his tongue hanging from his mouth, was lucky, Fossum said, because his owners, the Cordts family from San Antonio, noticed a lump on his leg and quickly sought treatment.

Osteosarcoma is the same malignant bone tumor seen in human children, said Mark Lenox, director of imaging for TIPS. It is the most common cancerous bone tumor in youth, and it usually appears during adolescence, tending to occur in the shin, thigh and upper arm.

“So we’re treating dogs and that’s good — they’re getting leading-edge treatment,” Lenox said. “But what we’re really targeting is how to cure this in children.”

Mitaplatin, a potent fusion of cisplatin and the orphan drug dichloroacetate

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Posted 11 Aug 2011 — by James Street
Category Cisplatin, DCA (Dichloroacetate), Dog Osteosarcoma, Glucose, Human osteosarcoma research, Molecular, Molecular Osteosarcoma Studies, Osteosarcoma, Warburg Hypothesis
  1. Shanta Dhara and
  2. Stephen J. Lipparda,b,1

+ Author Affiliations


  1. aDepartment of Chemistry and

  2. bKoch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139

  1. Contributed by Stephen J. Lippard, October 29, 2009 (received for review August 30, 2009)

Abstract

The unique glycolytic metabolism of most solid tumors, known as the Warburg effect, is associated with resistance to apoptosis that enables cancer cells to survive. Dichloroacetate (DCA) is an anticancer agent that can reverse the Warburg effect by inhibiting a key enzyme in cancer cells, pyruvate dehydrogenase kinase (PDK), that is required for the process. DCA is currently not approved for cancer treatment in the USA. Here, we present the synthesis, characterization, and anticancer properties of c,t,c-[Pt(NH3)2(O2CHCl2)2Cl2], mitaplatin, in which two DCA units are appended to the axial positions of a six-coordinate Pt(IV) center. The negative intracellular redox potential reduces the platinum to release cisplatin, a Pt(II) compound, and two equivalents of DCA. By a unique mechanism, mitaplatin thereby attacks both nuclear DNA with cisplatin and mitochondria with DCA selectively in cancer cells. The cytotoxicity of mitaplatin in a variety of cancer cell lines equals or exceeds that of all known Pt(IV) compounds and is comparable to that of cisplatin. Mitaplatin alters the mitochondrial membrane potential gradient (Δψm) of cancer cells, promoting apoptosis by releasing cytochrome c and translocating apoptosis inducing factor from mitochondria to the nucleus. Cisplatin formed upon cellular reduction of mitaplatin enters the nucleus and targets DNA to form 1,2-intrastrand d(GpG) cross-links characteristic of its own potency as an anticancer drug. These properties of mitaplatin are manifest in its ability to selectively kill cancer cells cocultured with normal fibroblasts and to partially overcome cisplatin resistance.

Normal cells typically use mitochondrial oxidative phosphorylation to metabolize glucose and switch over to glycolysis only when there is little or no oxygen, producing lactate as a byproduct. Cancer cells avidly consume glucose for energy by glycolysis to survive in the hypoxic environment of malignant lesions (1), a phenomenon known as the Warburg effect (2). The dependence of cancer cells on glycolysis comes not only from oxygen deprivation, but also partly from their inability to synthesize ATP in response to the mitochondrial membrane potential gradient (Δψm) (3). This unique glucose metabolic pathway of cancer cells has identified the mitochondrion as a prime target for cancer therapy (4–7). In addition, cancer cells develop the ability to avoid apoptosis by various pathways that ignore the command to commit cellular suicide (8, 9). Compounds that trigger apoptosis through selective action on mitochondrial target sites of cancer cells bypass defective upstream mechanisms and trigger apoptosis in tumor cells that are otherwise resistant (10).

Dichloroacetate (DCA) is used in humans to treat lactic acidosis (11). DCA inhibits the activity of pyruvate dehydrogenase kinase (PDK), thereby stimulating the mitochondrial enzyme pyruvate dehydrogenase (PDH). When turned off, PDH no longer converts pyruvate to acetyl-CoA required for mitochondrial respiration and glucose-dependent oxidative phosphorylation (12). DCA thus shifts cellular metabolism from glycolysis to glucose oxidation, decreasing Δψm (13) and helping to open mitochondrial transition pores (MTPs). This metabolic switch facilitates translocation of proapoptotic mediators like cytochrome c (cyt c) and apoptosis inducing factor (AIF), both of which stimulate apoptosis. DCA thereby drives cancer cells to commit suicide by apoptosis (13). Unlike most other anticancer agents, DCA does not appear to have any deleterious effect on normal cells. DCA reverses mitochondrial changes in a wide range of cancers, making malignant cells more vulnerable to normal cell death programs (14). Being an orphan drug, DCA is both nonpatentable and readily available, but it is not yet approved for use in cancer therapy (15). There is substantial preclinical evidence from both in vitro and in vivo models that DCA might be useful to treat cancer in humans, and a translation to early- phase clinical trials would be of interest (16–18). Funding for such trials would be a challenge because DCA is a generic drug. However, because it withdraws cancer cells from a state of apoptosis resistance, DCA is an attractive sensitizer that could be given concurrently with chemotherapy or radiation therapy. Alternatively, a formulation could be synthesized that incorporates DCA.

Platinum(II) compounds are used in 50% of all cancer therapies (19). Among these, cisplatin, carboplatin, and oxaliplatin have Food and Drug Administration approval and are in the clinic worldwide (20, 21). The use of platinum(II) drugs, cisplatin in particular, to treat malignancies is limited because of side effects and acquired resistance (22). Resistance can emerge from failure to execute apoptosis despite initiation of the apoptotic cascade caused by either the predominance of anti-apoptotic factors or defects in downstream effectors. Cisplatin resistance in ovarian carcinoma cells is associated with a reduced apoptotic response (23). To overcome tumor cell resistance and toxicity to normal tissues, we have been exploring strategies to target platinum constructs to cancer cells. Our tactic has been to employ substitutionally inert platinum(IV) compounds (24), which serve as prodrugs and release clinically effective levels of platinum(II) compounds, such as cisplatin, following cellular uptake (25–27). Appropriately designed platinum(IV) complexes are less likely to be deactivated before reaching their cancer cell destination target. The activity of platinum(IV) complexes generally involves reduction with loss of the axial ligands, affording an active platinum(II) complex that readily binds to DNA. Satraplatin is one such Trojan horse platinum(IV) compound that is currently under investigation for the treatment of patients with advanced prostate cancer (28).

We therefore designed a Pt(IV) compound (mitaplatin, 1) having two DCA moieties (Fig. 1) in the axial positions. We hypothesized that DCA released inside the cells by reduction of the platinum would simultaneously alter mitochondrial metabolism and deliver a dose of cisplatin (Fig. 1). Mitaplatin was thereby expected to have dual killing modes toward cancer cells, one in which cisplatin interacts with its key target, nuclear DNA, and the other, DCA released upon reduction, following a pathway to induce mitochondria-dependent apoptosis by mitochondrial membrane depolarization and efflux of proapoptotic mediators. Here, we describe the synthesis, characterization, and dual-action cell killing ability of mitaplatin as well as its remarkable ability to selectively destroy cancer cells in a coculture with normal fibroblasts.

Fig. 1.

Chemical structures and mechanism of action of mitaplatin (1). After crossing the plasma membrane, mitaplatin becomes reduced to release of the active drugs cisplatin and DCA. DCA inhibits mitochondrial PDK, which leads to PDH activation and increased glucose oxidation by promoting influx of acetyl-CoA into the mitochondria. DCA decreases the mitochondrial membrane potential (Δψm). Opening of the Δψm-sensitive mitochondrial transition pores (MTPs) leads to efflux of cyt c and AIF. Cisplatin formed in the reduction process interacts with its key target, nuclear DNA.

Results and Discussion

Synthesis and Characterization of Mitaplatin (1).

Mitaplatin (1), a formulation of DCA, was prepared by reaction of c,c,t-[Pt(NH3)2Cl2(OH)2] with dichloroacetic anhydride in >50% yield. Its formation was evidenced by disappearance of the O–H stretching band of the starting compound and the presence of a C=O stretch at 1651 cm−1 in the infrared spectra. The structure was confirmed by 1H, 13C, and 195Pt NMR spectroscopy, by HRMS, and by elemental analysis. ESI-HRMS (M–H) Calcd. = 554.8145, Found = 554.8138. 1H NMR (DMSO-d6) δ 7.95 (s, 2H), 6.52 (br, 6H); 13C NMR (DMSO-d6) δ 170.41, 65.27; 195Pt NMR (DMSO-d6): δ = 1205.28 ppm. Anal: Calcd for C4H8Cl6N2O4Pt: C, 8.64; H, 1.45; N, 5.04. Found: C, 8.13; H, 1.65; N, 4.88. Mitaplatin is redox-active and displays an irreversible reduction revealed by cyclic voltammetric analysis. The Pt(IV)/Pt(II) couple is near −0.173 V vs. Ag/AgCl at pH 7.4 and the value at pH 6.0 is −0.152 vs. Ag/AgCl. Voltammograms are given in Figs. S1 and S2. These reduction potentials indicate that mitaplatin will be readily reduced in cells. The cathodic reduction potential depends on the electron-withdrawing power and the bulkiness of the axial and auxiliary ligands. The low reduction potential of mitaplatin is influenced by the presence of chlorine atoms from the DCA molecules near the platinum center (29).

In Vitro Cellular Cytotoxicity Assays.

The ability of mitaplatin to promote cell death was evaluated by the MTT assay and the results were compared against those for cisplatin or DCA using NTera-2, HeLa, U2OS, A549, and MCF-7 cancer cells as well as MRC-5 normal fibroblasts (Fig. S3). Results are presented in Table 1. Mitaplatin has an IC50 value of 0.051 μM, comparable to that of cisplatin (IC50, 0.043 μM), in cisplatin-sensitive testicular NTera-2 cells and is more toxic than DCA alone. In U2OS osteosarcoma cells, cisplatin has an IC50 of 3.9 μM, whereas that of mitaplatin is 6.4 μM. Similarly, in HeLa cervical cancer cells, comparable IC50 values for mitaplatin and cisplatin were observed, 2.0 and 1.20 μM, respectively. Control experiments with the well known platinum(IV) compound c,c,t-[Pt(NH3)2Cl2(O2CCH3)2], revealed it to be several-fold less active than mitaplatin in all cells (Table S1). Mitaplatin was also established to have cytotoxicity comparable to that of cisplatin in the NCI/DTP 60 cell line growth inhibition assay, exceeding almost all known Pt(IV) compounds. This enhanced potency of mitaplatin is consistent with the expected dual killing mechanism.

Table 1.

Cell killing ability of mitaplatin

Mitaplatin Promotes Apoptosis in Cancer Cells.

To investigate the ability of mitaplatin to promote apoptosis in cancer cells by a mitochondrial-regulated mechanism, changes in the mitochondrial transmembrane potential (Δψm) of cancerous NTera-2 and healthy normal fibroblast cells before and after mitaplatin treatment were investigated by two assays. Mitochondrial attack is associated with a drop in Δψm. For this reason Δψm is an important parameter of mitochondrial function and has been used to monitor mitochondrial death. 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) is a lipophilic cationic dye which, depending on Δψm, accumulates as a green monomer in the cytoplasm or as red-emitting aggregates in hyperpolarized mitochondria of cancer cell (30). The negative charge established by the mitochondrial membrane potential allows the lipophilic dye, bearing a delocalized positive charge, to enter mitochondria where it accumulates. When a critical concentration is exceeded, J-aggregates form, which fluoresce red. In apoptotic cells, Δψm collapses, and JC-1 cannot accumulate in mitochondria. In these cells, JC-1 remains in the cytoplasm in a green fluorescent monomeric form. Control NTera-2 cells exhibited heterogeneous staining of the cytoplasm with both red and green fluorescence in the same cells (Fig. 2A). Treatment of these cells with mitaplatin for 4 h decreased the red fluorescence. Mitochondrial membrane depolarization was detected by a shift in fluorescence emission of JC-1 from red to green. There was no significant effect of mitaplatin on Δψm of normal fibroblasts or of cisplatin on Δψm of NTera-2 cells. The detailed results are given in Fig. S4.

Fig. 2.

Disruption of mitochondrial function and induction of apoptosis in cancer cells by mitaplatin. (A) Changes in the mitochondrial membrane potential as revealed by the JC-1 assay. Treatment with 100 μM mitaplatin dramatically caused the collapse of mitochondrial membrane potentials in NTera-2 cells. In live cells, JC-1 exists either as a green fluorescent monomer at depolarized membrane potentials (positive to −100 mV) or as an orange-red fluorescent J-aggregate at hyperpolarized membrane potentials (negative to −140 mV). The shift in membrane charge was observed by disappearance of fluorescent red-orange-stained mitochondria (large negative Δψm) and an increase in fluorescent green-stained mitochondria (loss of Δψm). (B) Reversal of mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM) assay. Mitaplatin significantly depolarized the NTera-2 cells but had no effect on the healthy normal fibroblast cells. Mitochondria were stained with mitotracker red. (C) Cytochrome c release visualized by fluorescence microscopy. Immunolocalization of cytochrome c (green) and mitochondrial morphology (red) shown in untreated NTera-2 cells and in mitaplatin treated NTera-2 cells. Cells were grown for 24 h on glass coverslips and treated with mitaplatin, fixed after treatment, and immunostained with anti-cytochrome c monoclonal antibodies. Mitochondria were stained with mitotracker red. (D) Translocation of AIF in mitaplatin treated cells. Staining of AIF (Ab) and nuclei (Hoechst) in NTera-2 cells before and after 12 h treatment with mitaplatin. Arrows indicate cells with particularly evident presence of AIF in the nucleus.

To investigate whether DCA released from mitaplatin can restore the hyperpolarization of cancer cells to the level of normal cells, we carried out a TMRM assay (31). All cancer cell lines have significantly more hyperpolarized Δψm compared to normal cells and therefore exhibit increased fluorescence of the Δψm-sensitive positive dye tetramethyl rhodamine methyl ester, TMRM. Incubation of the NTera-2 cells with mitaplatin for 48 h reversed the hyperpolarization and returned the Δψm to the level of normal cells (Fig. 2B). In contrast, mitaplatin did not alter the Δψm of the normal fibroblasts. Because dichloroacetate activates pyruvate dehydrogenase, which increases delivery of pyruvate into mitochondria, DCA released upon reduction of mitaplatin (Fig. 1) increased glucose oxidation, depolarizing the mitochondria and returning the membrane potential to levels of the noncancer cells.

Mitochondrial cyt c, which functions as an electron carrier in the respiratory chain, translocates to the cytosol in cells undergoing apoptosis, where it participates in activation of apoptotic proteins (32). The mechanism responsible for this process is unknown. Cyt c release from mitochondria is an early event in the apoptotic process induced by mitaplatin treatment in NTera-2 cells, as visualized by using a FITC-conjugated antibody for the protein and fluorescence microscopy. The cytosol from untreated cells showed no detectable cyt c (Fig. 2C). In contrast, cytosolic cyt c accumulated significantly after 4 h of treatment with mitaplatin.

AIF is a proapoptotic mitochondrial protein (33). Like cyt c, AIF is a bifunctional protein having both electron transfer and apoptogenic functions. AIF is released from mitochondria and translocated to nuclei, stimulating chromatin condensation and DNA fragmentation. We were interested to determine whether mitochondrial outer membrane permeabilization by mitaplatin induces apoptosis only by release of caspase-dependent factors, such as cyt c, or whether caspase-independent processes, such as that mediated by AIF, might be operative. We therefore investigated the location of AIF in the mitaplatin-treated cells. As shown in Fig. 2D, mitaplatin treatment led to translocation of AIF from the mitochondria to nuclei of NTera-2 cells.

To quantify mitaplatin-induced apoptosis in cancer cells, an annexin-V assay was performed by using flow cytometry. With this analysis, we determined the percentage of apoptotic cells at 48 h after exposure to mitaplatin, cisplatin, or DCA. Apoptosis was detected in cancerous U2OS, HeLa, and A549 cells with 10 μM mitaplatin and cisplatin. Cisplatin at 10 μM concentration evoked apoptosis in normal MRC-5 cells whereas mitaplatin did not produce any detectable apoptosis with these normal cells (Table 2).

Table 2.

Quantification of apoptosis induced by mitaplatin, cisplatin, and DCA using an annexin V assay

Visualization of Pt-1,2-d(GpG) Adduct Formation by Mitaplatin.

Because the anticancer activity of cisplatin derives from the formation of intrastrand 1,2-d(GpG) cross-links on nuclear DNA (34), we investigated whether cisplatin released by reduction of mitaplatin leads to this signature event by using a monoclonal antibody R-C18 (35) specific for this adduct (SI Text). After 12 h incubation of NTera-2 cells with mitaplatin, formation of 1,2-d(GpG) intrastrand cross-links was observed by antibody-derived green fluorescence in the nuclei of these cells (Fig. S5). These results confirm that mitaplatin has dual cell-killing modes involving DCA, which destroys mitochondrial function, and cisplatin, which simultaneously impedes DNA-mediated processes in the nucleus.

Selective Killing of Cancer Cells by Mitaplatin.

One of the main obstacles to cancer therapy is the inability to successfully target cancer cells, while not harming normal cells. Even when therapeutic agents are delivered locally to a primary tumor, systemic toxicities still arise. Modern medicine desperately needs anti-cancer molecules that kill cancer cells and leave healthy cells alone. Most cancer therapies today are very toxic to tumor and healthy cells alike, and the patient can succumb to treatment rather than the disease. Furthermore, cells develop a resistance to external agents, so chemotherapy may only work for a short time period. Because DCA has selective toxicity toward cancer cells by targeting PDK, we investigated whether mitaplatin would also display specificity for cancer. We therefore treated a coculture of normal fibroblasts and cancerous NTera-2 cells with mitaplatin, cisplatin, or a mixture of one equivalent of cisplatin and two equivalents of DCA, the stoichiometric composition released upon intracellular mitaplatin reduction. The morphology of these cells at different time points was examined by using bright field microscopy (Fig. 3A). The two types of cells in the coculture are clearly visible because of differences in their morphology. In the coculture, cisplatin and the mixture of cisplatin and DCA killed both the fibroblasts and NTera-2 cancer cells, whereas mitaplatin selectively killed the cancer cells. The results obtained in this study provide compelling evidence that mitaplatin can selectively kill cancer cells, leaving normal cells untouched.

Fig. 3.

Mitaplatin selectively induced apoptosis of human cancer cells and not normal cells. (A) Treatment of a coculture of normal fibroblast cells (elongated) and NTera-2 cells (round) with cisplatin, mitaplatin, and a mixture of one equivalent of cisplatin and two equivalents of DCA. (B) Selective killing of cancerous A549 (round) cells by mitaplatin in a coculture with normal MRC-5 (elongated) cells assayed using LIVE/DEAD staining. After mitaplatin or cisplatin exposure for 24 h, cells were stained with calcein AM (green fluorescence) and ethidium homodimer-1 (red fluorescence) to differentiate between live and dead cells, respectively.

To verify that preferential cancer cell killing occurs with mitaplatin, this study was further extended to a coculture of human lung cancer A549 cells and normal human lung fibroblasts MRC-5.

The selective killing of normal cells by mitaplatin was demonstrated by using a LIVE/DEAD viability assay, which allowed for the simultaneous determination of live and dead cells in a coculture by labeling live cells with calcein AM dye, which fluoresces only when cleaved by intracellular esterase enzymes, and ethidium heterodimer (EthD-1), which only enters dead cells with disrupted cell membranes (Fig. 3B). Fig. 3B confirms that, unlike cisplatin, a conventional chemotherapeutic agent, mitaplatin selectively induced cell death in human cancer A549 cells, but not in normal MRC-5 cells under the similar treatment conditions. To address whether the selective killing of cancer cells by mitaplatin might be a consequence of its selective uptake, we measured the nuclear and cytosolic concentrations of platinum by atomic absorption spectroscopy (AAS) after mitaplatin or cisplatin treatment of normal and cancer cells. Cytosolic and nuclear extracts were prepared from normal MRC-5 and cancerous A549 cells after incubation with 10 μM mitaplatin or cisplatin for 24 h. Platinum concentrations determined by AAS (Table S2) confirm the uptake of mitaplatin by both cells types.

Mitaplatin Action on Cisplatin-Resistant Cells.

Although very little is known about the effects of cisplatin on the mitochondria of tumor cells (36), a recent study showed that it might have direct impact on mitochondria in head and neck cancer (37). Mitochondrial defects are associated with the cisplatin resistance phenotype (38), and several hypotheses have been suggested to explain this observation. A more negative membrane potential might promote translocation of the active, cationic form of cisplatin from the cytoplasm to mitochondria, thus diminishing platination of nuclear DNA. This effect would suggest that a combination of cisplatin with a mitochondrial targeting moiety would be an attractive therapeutic strategy for attacking cisplatin-resistant tumors. We therefore studied a pair of cisplatin sensitive A2780 and resistant A2780/CP70 ovarian cancer cells (Table 1 and Fig. S6). As controls we used cisplatin and c,c,t-[Pt(NH3)2Cl2(O2CCH3)2]. The cells displayed a low level of resistance to mitaplatin (IC50 for A2780, 1.1 μM; IC50 for A2780/CP70, 3.34 μM) compared to cisplatin (corresponding IC50 values of 0.56 and 6.0 μM). Results for the A2780/CP70 cells indicate that DCA plays a role in making cisplatin-resistant cells susceptible toward mitaplatin treatment. A2780/CP70 cells were much more resistant to the control platinum(IV) compound c,c,t-[Pt(NH3)2Cl2(O2CCH3)2]. These data suggest mitaplatin as a promising candidate for further development in the treatment of cisplatin-resistant cells.

Summary.

In conclusion, mitaplatin displays a dual-killing mode that can only be effective in cancer cells. The platinum center interacts with its own target, nuclear DNA, and DCA released upon reduction attacks mitochondria. These results support the utility of mechanisms targeting cancer cell-specific pathways as an avenue for developing selective anticancer agents. Mitaplatin offers a formulation for future studies incorporating the orphan drug DCA to further its use in the clinic.

Materials and Methods

The complexes cis-[Pt(NH3)2Cl2] (39) and c,c,t-[Pt(NH3)2Cl2(OH)2] (40) were synthesized as described. Distilled water was purified by passage through a Millipore Milli-Q Biocel water purification system (18.2 MΩ) containing a 0.22-μm filter. Anti-cytochrome c (Ab-1) sheep polyclonal antibody was procured from Calbiochem. Alexa Fluor 488-labeled secondary antibody donkey anti-(sheep IgG) was obtained from Invitrogen for cytochrome c detection. For AIF detection, we used a rabbit polyclonal IgG antibody from Santa Cruz Biotechnology, Inc. Alexa Fluor 546-labeled secondary antibody goat anti-(rabbit IgG) was purchased from Invitrogen. The detection of the cisplatin 1,2-d(GpG) intrastrand adduct was carried out using a monoclonal adduct-specific antibody R-C18 which was kindly provided by Jürgen Thomale (University of Duisburg-Essen). FITC labeled secondary antibody rabbit anti-(rat Ig) was obtained from Invitrogen. Specific adhesion slides for immunofluoresecence were purchased from Squarix Biotechnology. JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbo-cyanine iodide) was obtained from Cayman Chemicals. 1H, 13C, and 195Pt NMR spectra were recorded on a Bruker AVANCE-400 NMR spectrometer with a Spectro Spin superconducting magnet in the Massachusetts Institute of Technology Department of Chemistry Instrumentation Facility (MIT DCIF). Atomic absorption spectroscopic measurements were taken on a Perkin-Elmer AAnalyst 300 spectrometer. HRMS analysis was carried out on a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometer in the MIT DCIF. Fluorescence imaging studies were performed with an Axiovert 200M inverted epifluorescence microscope (Zeiss) equipped with an EM-CCD digital camera C9100 (Hamamatsu). An X-Cite 120 metal-halide lamp (EXFO) was used as the light source. The microscope was operated with Volocity software (Improvision).

Synthesis of Mitaplatin c,c,t-[Pt(NH3)2Cl2(O2CCHCl2)2] (1).

To a solution of c,c,t-[Pt(NH3)2Cl2(OH)2] (0.2 g, 0.6 mmol) in DMF (5 mL) was added dichloroacetic anhydride (0.28 g, 1.5 mmol) and the reaction mixture was stirred at room temperature for 4 h. Diethyl ether was added to the mixture to precipitate a light yellow solid, which was washed several times with diethyl ether and dried. Mitaplatin (1) was isolated in 55% (0.29 g) yield. IR (KBr): νmax 3178, 3076, 3012, 1651, 1568, 1435, 1333, 1214, 1103, 1021, 819, 789, 723, 666, 582 cm−1; ESI-HRMS (M–H) Calcd. = 554.8145, Found = 554.8138. 1H NMR (DMSO-d6) δ 7.95 (s, 2H), 6.52 (br, 6H); 13C NMR (DMSO-d6) δ 170.41, 65.27; 195Pt NMR (DMSO-d6): δ = 1205.28 ppm. Anal: Calcd for C4H8Cl6N2O4Pt: C, 8.64; H, 1.45; N, 5.04. Found: C, 8.13; H, 1.65; N, 4.88.

Detection of Cyt c and AIF.

NTera-2 cells were seeded on microscope coverslips (1 cm) at a confluence of 1,600 cells per slip and incubated overnight at 37 °C in DMEM. The medium was changed and mitaplatin was added to a final concentration of 100 μM. The cells were incubated for 4, 12, or 24 h at 37 °C. The medium was then removed and the cells were incubated with fixing solution for 1 h at room temperature followed by three washes with PBS (pH 7.4). Cells were then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature for 1 h, then washed twice with PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min, followed by six washes with PBS. Cells were then rinsed with blocking buffer (PBS, 0.1% goat serum, 0.075% glycin), incubated for 1 h at 37 °C with the anti-cytochrome c [anti-cytochrome c (Ab-1) sheep pAb, Calbiochem] antibody or AIF [AIF (H-300), Santa Cruz Biotechnology Inc.] antibody, both diluted 1:50 in blocking buffer, washed twice with blocking buffer, and incubated at 37 °C with Alexa Fluor 488 donkey anti-(sheep IgG) (Invitrogen) antibody for cyt c release and Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) antibody for AIF (dilution 1:50 in blocking buffer) for 1 h. After two washes with blocking buffer and four washes with water, Mitrotacker Red for cyt c release and Hoechst bis-benzamide for AIF release were used to stain mitochondria and nuclei, respectively. Microscope coverslips were mounted on microscope slides using mounting solution for imaging.

JC-1 Assay.

GM61869 and NTera-2 cells were cultured on cover slips to a density of 1 × 106 cells/mL and incubated overnight at 37 °C. Cells were then treated with 100 μM mitaplatin for 4 and 48 h at 37 °C. A solution of JC-1 reagent (Cayman Chemicals; 10 μg/mL in DMEM) was added and incubation was carried out at 37 °C for 30 min. The cells were washed with PBS five times, fixed in 4% paraformaldehyde, and mounted onto glass slides using the procedure described above.

TMRM Assay.

Analysis of mitochondrial membrane potential (Δψm) was carried out by using TMRM. A similar procedure as mentioned above for the JC-1 assay was followed. Before fixing the cells, they were treated with 2 μM TMRM for 30 min at 37 °C.

LIVE/DEAD Assay.

In vitro selective killing was performed using the LIVE/DEAD Viability/Cytoxicity Assay (Molecular Probes). A549 and MRC-5 cells were cultured on sterile glass coverslips as subconfluent monolayers for 24 h at 37 °C in 5% CO2 and grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were then treated with 100 μM cisplatin or mitapltin for 24 h at 37 °C in 5% CO2. The cells were washed with Dulbecco’s PBS (D-PBS) to remove serum esterase activity generally present in serum-supplemented growth media before the assay. Calcein AM (4 mM in anhydrous dimethyl sulfoxide, DMSO) and EthD-1 (2 mM in DMSO/water, 1:4 vol/vol) were added to PBS (1:1,000 ratio) to produce a LIVE/DEAD working solution as recommended by the manufacturer. The samples were first washed in three changes of PBS and 100 μL LIVE/DEAD working solution was added on the coverslip and incubated at room temperature for 30 min. Subsequently, the samples were placed in PBS before being examined with a fluorescence microscope.

Annexin-V Assay.

Flow cytometry with a Vybrant Apoptosis Assay kit (annexin V conjugated to allophycocyanin, Invitrogen) was used to determine whether treatment specifically induces apoptosis. Briefly, 5 × 105 cells for each cell line were seeded into six-well tissue culture plates and incubated overnight to 60–70% confluence under standard growth conditions. Media for the cell lines were then replaced with fresh growth media with and without a 10 μM dose of cisplatin, mitaplatin, and a 20 μM dose of DCA. Treatment groups for each cell line were replicated three times. The cells were then incubated for 48 h at 37 °C and harvested with 0.25% trypsin-EDTA. Cells were washed with PBS and subsequently stained by annexin V as per the manufacturer’s protocol. Flow cytometry was performed on a BD LSR II flow cytometer (BD Biosciences) and data were analyzed on BD FACSDiva (BD Biosciences).

Acknowledgments

This work was supported by the National Cancer Institute Grant CA034992 (to S.J.L.) and the Koch Institute for Integrative Cancer Research (S.D.).

Footnotes

  • 1To whom correspondence should be addressed at:
    Department of Chemistry, Room 18–498, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307.

    E-mail: lippard@mit.edu

  • Author contributions: S.D. and S.J.L. designed research; S.D. performed research; S.D. and S.J.L. analyzed data; and S.D. and S.J.L. wrote the paper.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0912276106/DCSupplemental.

References

  1. ↵
    1. Gatenby RA,
    2. Gillies RJ

    (2004) Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4:891–899.

  2. ↵
    1. Warburg O

    (1956) On the origin of cancer cells. Science 123:309–314.

  3. ↵
    1. Samudio I,
    2. Fiegl M,
    3. Andreeff M

    (2009) Mitochondrial uncoupling and the Warburg effect: Molecular basis for the reprogramming of cancer cell metabolism. Cancer Res 69:2163–2166.

  4. ↵
    1. Larsson N-G,
    2. Luft R

    (1999) Revolution in mitochondrial medicine. FEBS Lett 455:199–202.

  5. ↵
    1. Schon EA,
    2. DiMauro S

    (2003) Medicinal and genetic approaches to the treatment of mitochondrial disease. Curr Med Chem 10:2523–2533.

  6. ↵
    1. Don AS,
    2. Hogg PJ

    (2004) Mitochondria as cancer drug targets. Trends Mol Med 10:372–378.

  7. ↵
    1. Spierings D,
    2. et al.

    (2005) Connected to death: The (unexpurgated) mitochondrial pathway of apoptosis. Science 310:66–67.

  8. ↵
    1. Hanahan D,
    2. Weinberg RA

    (2000) The hallmarks of cancer. Cell 100:57–70.

  9. ↵
    1. Kim J-W,
    2. Dang CV

    (2006) Cancer’s molecular sweet tooth and the Warburg effect. Cancer Res 66:8927–8930.

  10. ↵
    1. Xu R-h,
    2. et al.

    (2005) Inhibition of glycolysis in cancer cells: A novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia. Cancer Res 65:613–621.

  11. ↵
    1. Stacpoole PW,
    2. et al.

    (2006) Controlled clinical trial of dichloroacetate for treatment of congenital lactic acidosis in children. Pediatrics 117:1519–1531.

  12. ↵
    1. Stacpoole PW,
    2. Nagaraja NV,
    3. Hutson AD

    (2003) Efficacy of dichloroacetate as a lactate-lowering drug. J Clin Pharmacol 43:683–691.

  13. ↵
    1. Bonnet S,
    2. et al.

    (2007) A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell 11:37–51.

  14. ↵
    1. Stacpoole PW

    (1989) The pharmacology of dichloroacetate. Metab Clin Exp 38:1124–1144.

  15. ↵
    1. Pearson H

    (2007) Cancer patients opt for unapproved drug. Nature 446:474–475.

  16. ↵
    1. Cairns RA,
    2. Papandreou I,
    3. Sutphin PD,
    4. Denko NC

    (2007) Metabolic targeting of hypoxia and HIF1 in solid tumors can enhance cytotoxic chemotherapy. Proc Natl Acad Sci USA 104:9445–9450.

  17. ↵
    1. Cao W,
    2. et al.

    (2008) Dichloroacetate (DCA) sensitizes both wild-type and over expressing Bcl-2 prostate cancer cells in vitro to radiation. Prostate 68:1223–1231.

  18. ↵
    1. Wong JYY,
    2. Huggins GS,
    3. Debidda M,
    4. Munshi NC,
    5. De Vivo I

    (2008) Dichloroacetate induces apoptosis in endometrial cancer cells. Gynecol Oncol 109:394–402.

  19. ↵
    1. Galanski M,
    2. Jakupec MA,
    3. Keppler BK

    (2005) Update of the preclinical situation of anticancer platinum complexes: Novel design strategies and innovative analytical approaches. Curr Med Chem 12:2075–2094.

  20. ↵
    1. Rosenberg B,
    2. VanCamp L,
    3. Trosko JE,
    4. Mansour VH

    (1969) Platinum compounds: A new class of potent antitumor agents. Nature 222:385–386.

  21. ↵
    1. Wang D,
    2. Lippard SJ

    (2005) Cellular processing of platinum anticancer drugs. Nat Rev Drug Discovery 4:307–320.

  22. ↵
    1. Siddik ZH

    (2003) Cisplatin: Mode of cytotoxic action and molecular basis of resistance. Oncogene 22:7265–7279.

  23. ↵
    1. Perego P,
    2. et al.

    (1996) Association between cisplatin resistance and mutation of p53 gene and reduced Bax expression in ovarian carcinoma cell systems. Cancer Res 56:556–562.

  24. ↵
    1. Giandomenico CM,
    2. et al.

    (2002) Carboxylation of kinetically inert platinum(IV) hydroxy complexes. An entree into orally active platinum(IV) antitumor agents. Inorg Chem 34:1015–1021.

  25. ↵
    1. Dhar S,
    2. Gu FX,
    3. Langer R,
    4. Farokhzad OC,
    5. Lippard SJ

    (2008) Targeted delivery of cisplatin to prostate cancer cells by aptamer functionalized Pt(IV) prodrug-PLGA-PEG nanoparticles. Proc Natl Acad Sci USA 105:17356–17361.

  26. ↵
    1. Dhar S,
    2. Liu Z,
    3. Thomale J,
    4. Dai H,
    5. Lippard SJ

    (2008) Targeted single-wall carbon nanotube-mediated Pt(IV) prodrug delivery using folate as a homing device. J Am Chem Soc 130:11467–11476.

  27. ↵
    1. Mukhopadhyay S,
    2. et al.

    (2008) Conjugated platinum(IV)-peptide complexes for targeting angiogenic tumor vasculature. Bioconjugate Chem 19:39–49.

  28. ↵
    1. Choy H,
    2. Park C,
    3. Yao M

    (2008) Current status and future prospects for satraplatin, an oral platinum analogue. Clin Cancer Res 14:1633–1638.

  29. ↵
    1. Choi S,
    2. et al.

    (1998) Reduction and anticancer activity of platinum(IV) complexes. Inorg Chem 37:2500–2504.

  30. ↵
    1. Cossarizza A,
    2. Baccaranai-Contri M,
    3. Kalashnikova G,
    4. Franceschi C

    (1993) A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) Biochem Biophys Res Commun 197:40–45.

  31. ↵
    1. Floryk D,
    2. Houštĕk J

    (1999) Tetramethyl rhodamine methyl ester (TMRM) is suitable for cytofluorometric measurements of mitochondrial membrane potential in cells treated with digitonin. Biosci Rep 19:27–34.

  32. ↵
    1. Jiang X,
    2. Wang X

    (2004) Cytochrome C-mediated apoptosis. Annu Rev Biochem 73:87–106.

  33. ↵
    1. Joza N,
    2. et al.

    (2001) Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 410:549–554.

  34. ↵
    1. Jamieson ER,
    2. Lippard SJ

    (1999) Structure, recognition, and processing of cisplatin-DNA adducts. Chem Rev 99:2467–2498.

  35. ↵
    1. Liedert B,
    2. Pluim D,
    3. Schellens J,
    4. Thomale J

    (2006) Adduct-specific monoclonal antibodies for the measurement of cisplatin-induced DNA lesions in individual cell nuclei. Nucleic Acids Res 34:e47.

  36. ↵
    1. Park SY,
    2. et al.

    (2004) Resistance of mitochondrial DNA-depleted cells against cell death: Role of mitochondrial superoxide dismutase. J Biol Chem 279:7512–7520.

  37. ↵
    1. Cullen KJ,
    2. Yang Z,
    3. Schumaker L,
    4. Guo Z

    (2007) Mitochondria as a critical target of the chemotheraputic agent cisplatin in head and neck cancer. J Bioenerg Biomembr 39:43–50.

  38. ↵
    1. Harper M-E,
    2. et al.

    (2002) Characterization of a novel metabolic strategy used by drug-resistant tumor cells. FASEB J 16:1550–1557.

  39. ↵
    1. Dhara SC

    (1970) A rapid method for the synthesis of cis-[Pt(NH3)2Cl2] Indian J Chem 8:193–194.

  40. ↵
    1. Hall MD,
    2. et al.

    (2003) The cellular distribution and oxidation state of platinum(II) and platinum(IV) antitumor complexes in cancer cells. J Biol Inorg Chem 8:726–732.

UPenn Initiates Canine Osteosarcoma Study with Advaxis HER2

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Posted 08 Jul 2011 — by James Street
Category Dog Osteosarcoma, genetic, HER2/neu

press release

July 7, 2011, 2:45 p.m. EDT

 

PRINCETON, N.J., Jul 07, 2011 (BUSINESS WIRE) — Advaxis, Inc. ADXS +2.63% , a leader in developing the next generation of immunotherapies for cancer and infectious diseases, announces that the first dog has entered a dose-ranging in canine osteosarcoma at the University of Pennsylvania School of Veterinary Medicine.

Canine Osteosarcoma is a cancer of long (leg) bones that is a leading killer of large dogs over the age of 10 years. Standard treatment is amputation immediately after diagnosis, followed by chemotherapy. Invariably, however, the cancer metastasizes to the lungs. With chemotherapy, dogs survive about 18 months compared to 6-12 months, without treatment. The HER2 antigen is believed to be present in up to 50% of osteosarcoma. ADXS-HER2 creates an immune attack on cells expressing this antigen and has been developed to treat human breast cancer. The Company plans to file an IND later this year for this indication.

In 2010, Advaxis contracted with the University of Pennsylvania School of Veterinary Medicine to conduct a canine clinical program to determine the safety and efficacy of ADXS-HER2 in osteosarcoma. Positive results may lead to research in humans, as well.

“There is an especially high unmet need for safe, effective and reasonably priced cancer therapy in the companion animal market,” commented Thomas A Moore, Chairman and CEO of Advaxis, Inc. “This early study gets us started.”

About the Canine Osteosarcoma Trial

The study will be under the direction of Dr. Nicola Mason, an assistant professor at the University of Pennsylvania School of Veterinary Medicine. Only dogs with a histological diagnosis of osteosarcoma and evidence of expression of HER2/neu by malignant cells will be eligible for enrollment.

All dogs will receive 4 weeks of carboplatin therapy. Four weeks after the last carboplatin dose, dogs will receive ADXS-HER2 once every three weeks for a total of 3 doses. Group 1 (3 dogs) will receive 1×10(8) CFU per dose, Group 2 (3 dogs) will each receive 5×10(8) CFU per dose and Group 3 (3 dogs) will receive 1×10(9) CFU per dose. Additional dogs may be added to a Group to gather more data should if a potentially dose limiting toxicities, be observed. Therefore 9-18 dogs may be treated in the initial study.

About the University of Pennsylvania School of Veterinary Medicine

Penn’s School of Veterinary Medicine is one of the world’s premier veterinary schools. Founded in 1884, the School was built on the concept of Many Species, One Medicine(TM). The birthplace of veterinary specialties, the School serves a distinctly diverse array of animal patients, from pets to horses to farm animals at our two campuses. In Philadelphia, on Penn’s campus, are the Matthew J. Ryan Veterinary Hospital for companion animals, as well as classrooms, laboratories and the School’s administrative offices. The large-animal facility, New Bolton Center, in Kennett Square, Pa., encompasses hospital facilities for the care of horses and food animals as well as diagnostic laboratories serving the agriculture industry. The School has successfully integrated scholarship and scientific discovery with all aspects of veterinary medical education.

About Advaxis Incorporated

Advaxis is a biotechnology company developing proprietary, live but attenuated Listeria monocytogenes (Listeria) vaccines that deliver engineered tumor antigens, which stimulate multiple, simultaneous immunological mechanisms to fight cancer. Today, the Company has fifteen (15) distinct, cancer-fighting constructs in various stages of development, directly and through strategic collaborations with such recognized sites of excellence as the City of Hope, the Roswell Park Cancer Institute, the National Cancer Institute, the University of Pittsburgh, Cancer Research — UK, the University of British Columbia and the Department of Homeland Security. Please visit the Company’s portals: advaxis.com | facebook | twitter | LinkedIn

Forward-Looking Statements

Certain statements contained in this press release are forward-looking statements that involve risks and uncertainties. The statements contained herein that are not purely historical are forward looking statements within the meaning of Section 27A of the Securities Act of 1933, as amended, and Section 21E of the Securities Exchange Act of 1934, as amended. Forward-looking statements deal with the Company’s current plans, intentions, beliefs and expectations and statements of future economic performance. Forward-looking statements involve known and unknown risks and uncertainties that may cause the Company’s actual results in future periods to differ materially from what is currently anticipated. Factors that could cause or contribute to such differences include those discussed from time to time in reports filed by the Company with the Securities and Exchange Commission. The Company cannot guarantee its future results, levels of activity, performance or achievements.

SOURCE: Advaxis Incorporated

        Advaxis Incorporated
        Conrad F. Mir, 609-452-9813
        Executive Director
        mir@advaxis.com
        or
        Advaxis Incorporated
        Diana Moore
        Analyst
        dmoore@advaxis.com

Veterinary oncology chief named chairman of small animal clinical sciences

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Posted 05 Jul 2011 — by James Street
Category Cat osteosarcoma, Dog Osteosarcoma, genetic, vaccine
Filed under Announcements, InsideUF (Campus), Top Stories on Tuesday, July 5, 2011.

GAINESVILLE, Fla. — Rowan Milner, the Hill’s Associate Professor of Oncology at the University of Florida College of Veterinary Medicine, has been named the new chairman of the college’s department of small animal clinical sciences following a national search.

Milner, who also serves as chief of the oncology service for the UF Veterinary Hospitals, will succeed Colin Burrows in the position following Burrows’ retirement after nearly 30 years of service. Milner’s appointment was effective July 1.

“As chair, Dr. Milner will assume overall responsibilities for faculty recruitment, mentoring and promotion,” said Glen Hoffsis, the college’s dean. “He will also be responsible for budget management, leadership in research and veterinary and graduate student education.”

Milner will work closely with the hospital’s chief of staff to continue provide high-quality clinical service to the nearly 20,000 small animal patients that are treated annually at UF.

“Dr. Milner also will work with the scientific community of the Health Science Center, practicing veterinarians from Florida and other constituents of the college and our hospital,” Hoffsis said.

Dually board-certified in veterinary internal medicine and veterinary oncology, Milner received his early academic training from the University of Pretoria in South Africa. His research interests include osteosarcoma, melanoma vaccine, stereotactic radiosurgery, targeted radiotherapy and tumor suppressor genes.

He joined UF’s faculty in 2001 and has twice received Clinician of the Year awards from UF veterinary students. In recognition of his development of a promising new melanoma vaccine and for other research, Milner was named Clinical Researcher of the Year by the Florida Kennel Club in 2007. In 2011, he won the Pfizer Award for Research Excellence and in 2009 he received a faculty enhancement opportunity award from the Office of the Provost at UF.

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Credits

Contact
Sarah Carey, careysk@ufl.edu, 352-294-4242
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