Radical Ideas

By Sver Aune

Medical scientists at MUSC are uncovering new insights about how antioxidants work—details that are helping revive enthusiasm for their use in fighting cancer.

Oxygen, essential for life, can be toxic. Oxygen-centered free radicals are routinely synthesized and recycled as a normal part of cellular metabolism. Oxidative stress—when these highly reactive molecules tax a cell’s antioxidant systems—is a fundamental feature of many cancers.

Evolutionary theory states that, as oxygen became more abundant on primordial Earth, respiring single-celled organisms developed antioxidant systems to defend their DNA against free radicals formed from oxygen. Today, antioxidant enzymes are abundant in every cell type from bacteria to man. Conversely, cells produce low levels of oxygen-based free radicals to regulate metabolism and, when needed, to destroy invading viruses and bacteria. Maintaining the balance between oxidants and the antioxidant molecules that reduce them—redox balance—allows the body to exploit oxygen’s dual nature as both a sustainer of life and a lethal weapon against pathogens.

Of great importance to cancer biologists is the oxidant milieu surrounding a cancer cell, which is vastly different from the environs of a healthy cell. Similar to healthy cells, cancer cells can secrete toxic levels of oxidants to neighboring cells. This is a feature in certain breast and lung tumors, which deploy hydrogen peroxide to surrounding epithelial cells to push them toward a cancer phenotype. The major difference in many cancer cells is that they adapt to their own oxidative stress, allowing them to resist the lower levels of oxidants that healthy cells release in efforts to destroy them.

Kenneth D. Tew, Ph.D., DSc, Chair of the Department of Cell and Molecular Pharmacology at MUSC, explains the challenge of redox balance in cancer.

“When you are metabolizing more—which cancer cells do—you are essentially producing more oxidative stress, more oxygen byproducts,” said Tew. “Therefore, you need to generate a different redox homeostasis in the cell to counteract that.”

Understandably, the idea of using antioxidants as therapy in cancer retains promise as a means to restore redox balance. But can antioxidants help prevent cancer? And once cancer appears, can antioxidant therapies be developed to help fight it?

In the 1980s, high levels of oxidants were first observed on the molecular level in tumor cell lines. Interest in using dietary antioxidants as anti-cancer agents began to spread, spurring clinical trials.

There have been a handful of large randomized controlled trials designed to measure cancer risk when taking dietary antioxidant supplements prophylactically. Most showed that antioxidant supplements usually do not hurt, but they do not help either. In the Linxian General Population Nutrition Intervention Trial of gastric and esophageal cancer risk in the 1990s, healthy Chinese men and women took a daily combination of beta-carotene (15 mg), vitamin E (30 mg), and selenium (50 ug) for five years.¹ In the ten years following, there was no change in the risk of people developing or dying from either type of cancer.² In the Physicians’ Health Studies I and II conducted in the U.S. throughout the 1980s, 1990s, and 2000s, neither beta-carotene supplements (50 mg every other day) nor a combination of vitamins C (500 mg daily) and E (400 IU every other day) changed overall cancer risks—this was regardless of whether the physicians smoked.^3,4 The Women’s Health Study of women over 45 taking beta-carotene (50 mg every other day) or vitamin E (600 IU every other day) revealed the same.^5,6 The international HOPE-TOO trial in patients with heart disease or diabetes showed no change in cancer mortality with daily vitamin E supplementation (400 IU).⁷

In the SU.VI.MAX study, another large trial out of France, the results were mixed. A daily supplement cocktail of vitamins C (120 mg) and E (30 mg), beta-carotene (6 mg), selenium (100 ug), and zinc (20 mg) taken for roughly eight years actually increased women’s skin cancer risk but reduced men’s overall cancer risk.⁸ Five years later, both of these effects disappeared.⁹

In some people, certain antioxidant supplements should perhaps be avoided. In the Carotene and Retinol Efficacy Trial in the U.S., people with an increased risk of lung cancer due to smoking or asbestos exposure developed lung cancer more often and had overall reduced life spans when they took daily beta-carotene (15 mg) with vitamin A (25,000 IU).¹⁰ Similarly, in the ATBC study in Finland, smokers who took beta-carotene (20 mg per day) for five years contracted lung cancer more often than those who did not.¹¹ In another U.S. study, the Selenium and Vitamin E Cancer Prevention Trial, men over 50 taking daily vitamin E (400 IU) for five years developed prostate cancer 17 percent more often than those on placebo.¹²

Overall, antioxidants, at least in purified supplement form, do not seem to prevent cancer. And as much as smoking is a serious health risk, certain vitamins might increase that risk.

To date, the potential benefits of using antioxidants to prevent cancer seem equivocal. However, novel insights are enabling researchers at MUSC to target the underlying components of redox imbalance in certain cancers. New knowledge of these targets is reviving the promise of using antioxidants as adjuvant agents that could protect healthy tissue from the oxidative stresses of radiation and chemotherapy.

When considering how to treat a cancer, Tew always refers back to redox balance within normal cells. Curiously, healthy cells use sulfur, oxygen’s neighbor on the periodic table, to keep oxidants in check. The ubiquitous cellular antioxidant glutathione forms sulfur-sulfur bonds with proteins, in a protein modification process called glutathionylation, to help them fold so they can function properly. In moments of oxidative stress—during radiation, for example—glutathionylation effectively shields proteins from being irretrievably damaged by oxidants.

“Life developed through a process of using oxygen and sulfur as signaling molecules,” said Tew. “It’s this yin and yang effect of oxygen and sulfur that works very well for the life forms that we are.”

Tew suspects drugs that aid glutathionylation will have significant biological importance in cancer. Ezatiostat hydrochloride (Telintra®; Mabvax Therapeutics Holdings, Inc; San Diego, CA) is a drug designed as a glutathione analog and is in phase 2 clinical trials for myelodysplastic syndrome, a precursor disease of leukemia. Ezatiostat inhibits a particular enzyme complex that regulates glutathionylation, and as a consequence, affects downstream phosphorylation pathways that control cancer cell growth. Tew is working to further determine ezatiostat’s efficacy in myelodysplastic syndrome, with the goal to include it in further clinical trials at MUSC.

Tew is also performing a clinical trial with David T. Marshall, M.D., a radiation oncologist at the MUSC Hollings Cancer Center, to track glutathionylation in the blood of cancer patients who have received radiation. The goal in patients is to determine how much treatment is too much and to stay on the safe side of that line.

“Radiation and drug treatment stimulate modifications of proteins because they are trying hard to achieve redox homeostasis,” said Tew. “We can use these proteins as potential biomarkers of response to either drugs or radiation.”

Tew and Marshall’s research group has already identified several candidate biomarkers in the cheek cells of healthy volunteers who used a hydrogen peroxide mouthwash that safely mimics the oxidative stress of radiation. Initially, changes in these biomarkers will be tracked during tests of novel anti-cancer drugs. The next step will be to track those same biomarkers in patients who are receiving radiotherapy.

If antioxidants are to live up to their promise as anti-cancer agents, better delivery models are needed. Bioengineer Ann-Marie Broome Ph.D., MBA, believes that clinical trials with antioxidants have been disappointing thus far because the body rapidly metabolizes and clears them to maintain redox balance. To evade these natural processes, the Broome laboratory, including physical chemist Suraj Dixit, Ph.D., and organic chemist Yu-Lin Jiang, Ph.D., makes tiny drug delivery devices called nanoparticles that, according to Broome, “package a drug and put the entire construct in stealth mode.”

Their objective is to bypass the body’s powerful drug metabolism systems and deliver drugs with antioxidants directly to the tumor.

“Although we have very good drugs to treat cancer, many deleterious side effects accompany their use because we typically treat the whole body rather than a specific location,” said Broome.

Chemotherapy doses are higher and more expensive than they should be, said Broome, because only a fraction of the drug reaches the target. This is especially true in glioblastoma since the chemotherapeutic agent must cross the blood-brain barrier to reach the tumor. For example, only about 0.3 percent of intravenous temozolomide, the current standard of care, reaches the tumor. Even after surgery and chemotherapy, glioblastoma has a 100 percent recurrence rate.

Broome partners with colleague Amy Lee Bredlau, M.D., Director of the Pediatric Brain Tumor Program, to procure samples from surgically resected glioblastoma. Bredlau is working to identify vulnerabilities in glioblastoma and to understand the genetic backgrounds in which the tumors appear. They have learned that, similar to certain breast and lung cancers, these tumors thrive in a low-oxygen environment and produce cytotoxic levels of hydrogen peroxide. For the aggressive tumors, hydrogen peroxide provides the “second hit”—a chemical signal that keeps them growing and progressing.

Drug delivery devices are not just chemically synthesized, they are built. A typical nanoparticle will have an outer coating made to disguise it from the immune system, along with the appropriate chemotherapy drug packaged in the center, and a targeting molecule that recognizes some specific feature of the tumor. An outer cloak incorporates an antioxidant coating made of N-acetylcysteine or resveratrol (the antioxidant found in red wine) that can “mop up” the tide of hydrogen peroxide surrounding a tumor. In these cases, the antioxidant coatings are both the package and part of the therapy.

There are challenges in scaling up production of nanotherapies. The particles are tiny—small enough to cross the blood-brain barrier—and much finesse and effort are required to produce an adequate supply for experiments. But results in preclinical studies are encouraging. Broome and Bredlau have raised the temozolomide concentration in the brain from 0.3 percent to 3 percent of the original injected drug—a ten-fold increase. Broome hopes production will take hold on an industrial level as they augment their experimental techniques.

At MUSC, the anti-cancer promise of antioxidants is clear. Since redox balance is crucial to the survival of every cell, the field is both challenging and potentially very rewarding. The fundamental lessons learned in glioblastoma and myelodysplastic syndrome could be extended to many conditions in which redox status is disrupted—heart and lung disease, stroke, diabetes, even other types of cancer. After all, oxygen, while toxic, is also necessary for life.

References

¹ Blot WJ, et al. J Natl Cancer Inst 1993;85(18):1483-1491.

² Qiao YL, et al. J Natl Cancer Inst 2009;101(7):507-518.

³ Hennekens CH, et al.N Engl J Med 1996;334:1145-1149.

⁴ Gaziano JM, et al. JAMA 2009;301(1):52-62.

⁵ Lee IM, et al.J Natl Cancer Inst 1999;91(24):2102-2106.

⁶ Lee IM, et al. JAMA  2005;294(1):56-65.

⁷ Lonn E, et al. JAMA 2005;293(11):1338-1347.

⁸ Hercberg S, et al.J Nutr 2007;137(9):2098-2105.

⁹ Hercberg S, et al. Int J Cancer 2010;127(8):1875-1881.

¹⁰ Omenn GS, et al. N Engl J Med 1996;334(18):1150-1155.

¹¹ Alpha-Tocopherol Beta Carotene Cancer Prevention Study Group. N Engl J Med.. 1994 Apr 14;330(15):1029-1035.

¹² Klein EA, et al. JAMA 2011;306(14):1549-1556.