Promising cancer therapy dismissed in ’70s earns second chance

By Jennifer Brown

The 20th-century chemist Linus Pauling is famous for advocating the powerful health benefits of vitamin C, including his assertion in the mid-1970s that massive amounts could kill cancer cells.

Working with Scottish surgeon Ewen Cameron, Pauling reported that cancer patients given high doses of vitamin C, or ascorbate, lived significantly longer than patients who didn’t get the treatment. The results were exciting, but when a team at the Mayo Clinic rigorously tested Pauling’s theory using randomized clinical trials, they found absolutely no survival benefit from vitamin C, dooming Pauling’s claim.

When a potential cancer therapy shows great promise in the lab only to fail dismally in the clinic, it rarely gets a second chance. For vitamin C, the wait was more than three decades before a meticulous scientist at the National Institutes of Health teamed with determined—some might say pigheaded—University of Iowa researchers who thoroughly understood the chemistry of vitamin C and believed its role as a cancer killer was worth reviving.

Free radical ideas in Iowa

Appreciating the potential power of vitamin C requires first an understanding of weaknesses in cancer cells—a theory advanced by Larry Oberley, PhD, who led the Free Radical and Radiation Biology Program at the UI from 1998 until his death in 2008. He turned Iowa into something of a mecca for free radical biologists.

Oberley’s research showed that in cancer cells there is an imbalance between free radicals—which can be damaging to biological tissues and molecules like DNA—and the antioxidant enzymes that keep free radicals under control in normal cells. He proposed that exploiting these fundamental flaws could lead to new ways to treat cancer.

“We know that tumor cells are different from normal cells,” says Joe Cullen (’86 MD, ’91 R), UI professor of surgery. “One of the differences we focus on and try to use is that cancer cells make more reactive oxygen species than normal cells. We think using adjuvant treatments that increase the level of ROS in cancer cells even more will push them over the edge, increasing their vulnerability to conventional chemotherapies and radiation.”

When NIH scientist Mark Levine, PhD, discovered a previously unrecognized difference between Cameron and Pauling’s efforts and the Mayo clinical trials, it suggested that pharmacologic ascorbate might be a potential candidate for tipping the balance in cancer cells.

Levine showed in the 1990s that orally ingested vitamin C is subject to very tight control, which maintains plasma levels in the micromolar range even when people consume very large quantities. He later realized that delivering vitamin C intravenously would bypass this tight regulation and could produce much higher concentrations—millimolar levels—of ascorbate in plasma.

This striking difference provided a potential explanation for the opposing results obtained in the Cameron-Pauling experiments, where the patients received both oral and intravenous (IV) ascorbate, and in the Mayo clinical trials where patients received the vitamin C orally.

Levine turned to vitamin C chemistry expert Garry Buettner (’69 MS, ’76 PhD), UI professor of radiation oncology and a close collaborator of Oberley. Buettner’s research focuses on oxidation and reduction reactions involving ascorbate and the role these reactions play in health and disease. He understood that vitamin C is generally viewed as an antioxidant, but under certain circumstances ascorbate can also function as a pro-oxidant, generating excess ROS and increasing oxidative stress.

Levine and Buettner conducted experiments in human cancer cells and animal models of cancer that unequivocally showed millimolar doses of ascorbate can selectively kill cancer cells but are harmless to normal cells. Importantly, they showed that this effect was mediated by ascorbate’s pro-oxidant action—at the high concentrations achieved in the experiments, ascorbate acts as a prodrug, delivering damaging hydrogen peroxide to tumor cells.

Second chance for C

Buettner and Levine saw an opportunity to revisit vitamin C’s role in treating cancer. But given its failure in the ’70s, they knew it would be a tough sell.

“There was still resistance from people who didn’t look at the new information. But our studies gave us a new way to think about mechanism and that gave us ways to think about how to make it work better,” Buettner says.

Given the UI’s expertise in free radical biology, Buettner felt that UI cancer physicians were perhaps more likely than most to be receptive to their idea, so he had Levine present their findings to the UI oncologists.

Among the “takers” was Cullen, who was interested in pancreatic cancer. Cullen and his team set up lab experiments to test the cancer-killing potential of vitamin C for themselves.

“This worked better than anything we ever tried,” Cullen says. “If you gave the cancer cells 10-to-20-millimolar ascorbate for just an hour, which we can easily achieve in humans, nothing survived. In contrast, normal cells were unaffected by the same treatment.”

The researchers also showed that high-dose ascorbate therapy inhibited tumor growth in a mouse model of pancreatic cancer. However, the treatment alone did not destroy the tumors in the animal.

“That result informed our thinking right away,” says Buettner. “We realized we would likely need to combine the ascorbate with other therapies.”

Because of ethical considerations, new cancer therapies are always tested in conjunction with the standard-of-care treatment. So in 2009, Cullen and Buettner, in consultation with Levine at NIH, initiated the phase 1 PACMAN (Pharmacological Ascorbate with Gemcitabine for the Control of Metastatic and Node-Positive Pancreatic Cancer) clinical trial to test the safety and tolerability of pharmacologic ascorbate combined with the standard chemotherapy agent gemcitabine in patients with stage 4 pancreatic cancer.

Dotting the i’s, crossing the t’s

The researchers were acutely aware of the need to be precise and get everything right this time. They used rigorous quality control, ensuring the infused ascorbate solution did not become oxidized and remained bioactive; monitoring patients’ plasma levels of ascorbate before and after infusion; increasing the dosage gradually to ensure that patients could tolerate the therapy; and infusing the solution slowly to avoid problems due to the high salt load.

In discussing the new trial with Cullen, Buettner remembers saying, “We have to make sure that every ‘i’ is dotted and ‘t’ is crossed, because if something goes wrong, this is dead for another generation.”

The attention to detail paid off. The PACMAN study successfully demonstrated that IV ascorbate is safe and well-tolerated by patients, and of the nine patients who received two cycles of ascorbate, overall survival was 14 months compared to an average of six months commonly seen for people with this late-stage, aggressive cancer.

“This was a phase 1 trial so we can’t judge efficacy from the results, but it is promising,” Cullen says.

The success of the PACMAN trial for vitamin C, particularly the ease with which patients tolerated the treatment, and data from the UI trial and others suggesting that quality of life also improved in patients on high-dose IV ascorbate has caused a surge in interest among UI clinicians.

Two more phase 1 clinical trials have started: one for locally advanced, stage 3 pancreatic cancer, which combines IV ascorbate with radiation and chemotherapy; and another trial that combines IV ascorbate with chemotherapy and radiation for patients with glioblastoma multiform (GBM), the most aggressive form of brain cancer. If funding can be secured, UI clinicians also hope to start a phase 1 trial for stage 4 prostate cancer.

In addition, positive lab results with melanoma and sarcoma cells have raised hopes of initiating phase 1 trials for these cancers, too.

Finally, the researchers are on the verge of starting the first phase 2 clinical trial of pharmacologic ascorbate combined with chemotherapy for patients with stage 4 lung cancer. The protocol has already been shown to be safe in a phase 1 trial for ovarian cancer conducted at the University of Kansas.

Ketogenic diet in trials as well

UI researchers also are testing the ketogenic diet as another approach to exploit cancer cells’ faulty metabolism—and improve responses to conventional chemoradiation therapies—by selectively enhancing oxidative stress.

UI researcher Douglas Spitz (’84 PhD), professor of radiation oncology, had previously shown that glucose deprivation preferentially kills cancer cells compared to normal cells by exploiting the oxidative metabolic pathways that ascorbate targets. A ketogenic diet, which is a high-fat, low-carbohydrate regimen that has been used safely for years to treat epilepsy, deprives cancer cells of glucose and forces them to metabolize fats in their mitochondria. This causes oxidative stress, which makes cancer cells more susceptible to chemotherapy and radiation.

“Cancer cells are more stressed because they have more oxidants,” explains Bryan Allen (’08 MD, ’08 PhD, ’13 R), UI assistant professor of radiation oncology. “We are just trying to tip the balance with the ketogenic diet and ascorbate, so that the cancer cells, but not normal cells, become overwhelmed with the free radicals and oxidants. Once we saw we could do it in one or two cancer sites, we just started to do it in multiple sites.”

There are three phase 1 clinical trials under way at the UI to test the ketogenic diet in conjunction with standard chemotherapy and radiation for locally advanced, non-small-cell lung cancer, locally advanced pancreatic cancer, and advanced head and neck cancer. Based on strong lab results, Spitz and Allen hope to start a phase 2 trial of the ketogenic diet in the near future.

As clinical testing moves forward on both the ketogenic diet and high-dose vitamin C for multiple cancer types, Spitz is quick to credit Allen’s enthusiasm as a catalyst for advancing preclinical findings into testing in human trials.

“The excitement is so high among patients who participate in these trials,” Allen says. “They often bring family members along and I talk to them and show them pictures from our lab work showing fluorescent tumor cells in mice and how they shrink in the mice being treated with the high-dose vitamin C. These patients are really participating in their care and also helping future patients down the line with what we learn.”

Exploiting cancer’s flaws

By Jennifer Brown

Cancer cells have a reputation as “super cells”—indestructible and capable of unlimited growth. But scientists in the University of Iowa Free Radical and Radiation Biology Program take a different view.

They see cancer cells as intrinsically flawed in their ability to control reactive oxygen species (ROS)—such as superoxide and hydrogen peroxide—which makes cancer cells vulnerable to therapies that increase oxidative stress.

The problem for cancer cells is believed to be due to abnormalities in their mitochondrial electron transport chain, the complex metabolic machinery that converts oxygen and food into energy and biosynthetic power.

“Basically you eat food to get high-energy electrons. You breathe oxygen to get rid of these electrons after extracting useful energy. You age and die because of the inefficiencies between these two processes,” says Douglas Spitz, PhD, UI professor of radiation oncology.

During metabolism, high-energy electrons are plucked from foodstuffs in oxidation reactions. These electrons are then shuttled down the electron transport chain in cells’ mitochondria, to generate energy and “reducing power” to drive biosynthesis. At the end of the ETCs, oxygen molecules each accept four electrons to form two water molecules, which is also essential for biological life.

“It’s almost a perfect system,” Spitz adds. “Almost all of the oxygen that we inhale proceeds through this four-electron reduction scheme. But there is a slight inefficiency. Sometimes oxygen can diffuse away from those reactions with only one or two of those electrons, instead of four, and these oxygen molecules are potentially harmful reactive oxygen species.”

Through subsequent electron transfer reactions, that “slight inefficiency” can generate highly reactive molecules such as hydroxyl radicals, which Spitz calls “the most powerful oxidant in biology.”

Experimental evidence suggests that in cancer cells, abnormalities in this mitochondrial oxidative metabolism lead to even greater inefficiencies and, consequently, increased steady-state levels of ROS that can result in chronic oxidative stress.

This framework for understanding cancer cell metabolism provides a rationale for using interventions like pharmacological ascorbate and a ketogenic diet (high-fat, low-carb), which take advantage of these defects in cancer cells’ metabolism to further increase the levels of ROS in cancer cells and increase their susceptibility to chemotherapies and radiation.

“What I tell my patients is that vitamin C (and a ketogenic diet) is selectively increasing the oxidative stress in cancer cells—all those free radicals that cause damage to proteins and DNA,” says Bryan Allen, MD, PhD, assistant professor of radiation oncology. “Then we hit the cells again with radiation and chemotherapy, making those standard-of-care treatments that much more effective. Conversely, vitamin C does not seem to stress normal cells in the same way and that’s how we are able to increase the effectiveness of the chemotherapy and radiation.”