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Nitric Oxide:

Just say NO to Cancer and Much More

Nitric oxide (NO)—a simple molecule consisting of one nitrogen atom and one oxygen atom—is everywhere. Blood vessels use it to relax, neurons use it to communicate, and innate immune cells use it to kill dangerous invaders. NO researchers won the Nobel Prize in 1998, barely a decade after its identification as the biological activity known as endothelial-derived relaxing factor (EDRF). As the initially controversial evidence began to accumulate that NO is a key player in several biological processes, Larry Keefer, Ph.D., Chief of CCR’s Laboratory of Comparative Carcinogenesis, was ready with the tools to manipulate it for biomedical research. An organic chemist working on NO-related chemistry as a means to understand and ultimately prevent the carcinogenic effects of the nitrosamines found in a variety of foodstuffs, environmental sources, and manufacturing processes, Keefer was poised to jump into the NO fray with the first reliable chemical donor with which to study the effects of authentic NO in culture and in vivo. Since then, he has initiated several collaborations to develop and study agents that can selectively target NO’s power to destroy cancerous cells. His goal is to see one of these agents enter the clinic.

Photo shows Larry Keefer, Ph.D.
Larry Keefer, Ph.D. (Photo: R. Baer)

A volatile gas on its own, nitric oxide (NO) is produced where it is needed in the body, and it cannot be directly administered to most biological tissues. Instead, it must be released from other compounds, as in the case of nitroglycerin, which was used to treat heart pain a hundred years before the mechanism producing dilation of blood vessels was shown to be NO.

"My group is [almost entirely] chemists," said Keefer. "We know how to make compounds, make them pure, and design them with specific structural features. That’s been our forte. We try to interest collaborators who know how to do the rest of it." One of his most fruitful collaborations has been with University of Utah oncologist Paul Shami, M.D., to study the use of NO to fight cancer.

NO to Leukemia

Shami demonstrated several years ago that acute myeloid leukemia cells are particularly sensitive to NO toxicity at concentrations of a NO-releasing drug substantially lower than those that safely maintain normal healthy endothelial or liver cells.

"NO had long been known as a toxic air pollutant, cigarette smoke constituent, and precursor of carcinogenic nitrosamines. But as its numerous bioeffector roles attest, NO turns out to be essential for proper health just about everywhere in your body," explained Keefer. "So, evolution has provided our cells with ways to deal with its toxic potential." Shami’s leukemia cells had apparently lost some of that ability. This led him to the hypothesis that administering a NO-releasing drug into the general circulation might preferentially eliminate the NO-sensitive leukemia cells without collateral harm to normal tissues. This proved to be the case in a mouse xenograft model of leukemia—a research model in which human cancer cells are grafted under the skin of mice with impaired immune systems to prevent rejection of the foreign graft. The lead compound Shami identified, JS-K, cut the growth rate of the mice’s tumors in half without any apparent toxic effects; it also induced more necrotic cell death, relative to controls, in the tumor mass that remained.

The preclinical evidence is mounting for JS-K’s potential as a novel anticancer agent.

Having learned of Shami’s success with the in vivo leukemia model, Tanyel Kiziltepe, Ph.D., and Kenneth Anderson, M.D., at Harvard Medical School, demonstrated that JS-K also inhibits proliferation of human myeloma cells in vitro as well as in xenograft models. Because of JS-K’s cell specificity, the doses required to see an effect in mice did not, as expected, cause major changes in vascular tension. The researchers have also studied the mechanisms through which JS-K damages cancer cells and have found evidence for NO-induced DNA damage leading to apoptosis. "I can’t put together the whole story on the mechanism yet," noted Keefer. "You look at the structure and chemistry, and there are clearly other pathways by which the compound can be active." Nonetheless, the preclinical evidence is mounting for JS-K’s potential as a novel anticancer agent. In a paper published earlier this year in Leukemia Research, the team demonstrated that JS-K has a synergistic effect with the antileukemia drug cytarabine in inhibiting proliferation of leukemia cell lines. Shami, in the meantime, has founded a biotechnology company with a confidently optimistic name—JSK Therapeutics.

The efficacy of JS-K appears to extend beyond leukemia and multiple myeloma cells. Similar cytostatic effects have been observed in rodent liver and prostate cancer models. Keefer is also collaborating with Lucy Anderson, Ph.D., Head of the Cellular Pathogenesis Section at CCR, to study JS-K in the multiple human lung cancer cell lines that she and her colleagues have characterized for NO research. Working across the two laboratories, Research Associate Anna Maciag, Ph.D., has unpublished data demonstrating that JS-K is not only effective against lung cancer cells but that it also appears to have an even greater potency in lung cancer cells that have high levels of reactive oxygen species (ROS). "Now we’re talking about personalized medicine," commented Keefer. "If a tumor contains high levels of ROS, perhaps it will be an ideal candidate for our drug."

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