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Striking a Blow Against Cancer

Trapping the calcium in a compartment in the cell’s nucleus makes the tumor more vulnerable to radiation therapy

Steve Gschmeissner/Science Photo LibraryCancer cell of the mouth (in red), as seen through a scanning electron microscopeSteve Gschmeissner/Science Photo Library

Bombarding a tumor with X-rays can prevent it from spreading throughout the body. But it’s a delicate operation. Even though the radiation therapy eliminates most of the cancer cells by damaging their DNA and inducing their death, the few remaining can mutate to become immune to the effects of radiation and increase their capacity to reproduce. Thus, the tumor grows back, faster and more resistant, and the only option for physicians is to remove it by surgery. In the case of head and neck cancer, however, this last resort is often crippling. It is not aesthetically pleasing and compromises swallowing, speaking and even breathing.

Seeking a more effective way to combat this type of tumor, a multidisciplinary team of 18 researchers from Minas Gerais and São Paulo has developed a promising strategy to make tumor cells more sensitive to radiation. The results of the research, published in May of this year in the Journal of Cancer Science and Therapy and still in the preclinical phase, suggest that radiation therapy followed by a procedure that prevents the free movement of calcium ions in the cell’s nucleus can cut in half the amount of radiation exposure and dramatically decrease the risk of the tumor’s recurrence.

Under the supervision of Maria de Fátima Leite, a cell biologist at the Federal University of Minas Gerais (UFMG), and Olindo Assis Martins Filho, an immunologist at the René Rachou Research Center, of the Oswaldo Cruz Foundation in the city of Belo Horizonte, Lidia Maria Andrade, a researcher, came up with this strategy. While still a dental student, Andrade had the opportunity to closely monitor the suffering of patients with cancers of the head and neck. Caused primarily by smoking and alcohol consumption, it is the eighth leading cause of cancer deaths in the world. The National Cancer Institute estimates that oral cancer alone will affect more than 14,000 people this year in Brazil. Doctor João Victor Salvajoli, a specialist in radiation therapy at the Hospital Alemão Oswaldo Cruz (Oswaldo Cruz German Hospital), in São Paulo, says that radiation therapy is often combined with chemotherapy in the treatment of head and neck cancers, and the substances used in the process make cells more sensitive to radiation. Side effects include lesions on the skin and mucosa that can severely affect swallowing, taste and salivation. Furthermore, there is a high risk of recurrence of the disease.

Andrade, who was interested in finding something that would further decrease the resistance of head and neck cancer cells to radiation, sought out Leite in 2008. Her research in the last decade had focused on studying the role played by calcium ions of the cell’s nucleus in the proliferation of tumors. And so the researchers decided to investigate if the buffering of calcium flux would decrease the resistance of these cells to radiation.

Blocked channels
Calcium ions are key components of the machinery that drives cells. The greater or lesser concentration of calcium triggers or suppresses a number of vital processes, such as the activation of genes. Within the cells there is an organelle with several compartments that store calcium ions, which is known as the endoplasmic reticulum. The walls of this reticulum are covered with proteins, some of which act as channels. These are the so-called calcium channels, which remain closed most of the time and only open when a particular substance, inositol triphosphate, binds to them, which allows ions to be released into the cytoplasm. As needed, calcium ions are pumped back into the interior of the reticulum.

054-057_Calcio_201_novoIn 2003, in collaboration with Cornell and Yale University researchers in the United States, Leite, in an article in the journal Nature Cell Biology, described how control of the calcium in the cell’s nucleus is independent of the calcium located in the rest of the cell. The nucleus has another reservoir of these ions, which is called the nucleoplasmic reticulum. In 2007, with her Yale colleagues, Leite published a paper in the Journal of Biological Chemistry demonstrating that blocking 70% of the calcium released from the nucleoplasmic reticulum reduced the cell reproduction of a liver tumor implanted in mice. “The tumor grew absurdly less than normal,” says Leite. “It has been known for a long time that calcium participates in various stages of cell multiplication, the so-called cell cycle,” she adds. Blocking calcium interrupts the cell cycle and thus the cell is unable to divide.”

Calcium blocking was obtained by means of a genetic engineering technique both in the liver tumor and now in the cells of head and neck cancers cultured in the laboratory. With the aid of an artificial adenovirus, the researchers induced tumor cells to produce a protein within the nucleus that mimics the part of the calcium channels that binds to inositol triphosphate. With almost all inositol triphosphate neutralized by these proteins, most of the actual calcium channels of the nucleoplasmic reticulum remain closed (see infographic). Without the required amount of calcium, tumor cells have decreased capacity to reproduce.

This procedure provided an additional advantage. Blocking calcium reduced only the proliferation of tumor cells. Healthy gum cells, when subjected to gene therapy, continued to reproduce normally. According to the researchers, healthy cells proved to be less sensitive to the retention of nuclear calcium. This finding suggests that a therapy based on this strategy would not damage the healthy tissue surrounding the tumor.

Realistic test
In tests, Andrade and the team of Jony Marques Geraldo, a physicist at São Francisco Radiation Therapy Institute in Belo Horizonte, irradiated cell cultures with X-rays, taking care that each received the same amount of radiation – the experiment could have had misleading results if some cells received more or less radiation than others. They were also careful to try to reproduce the actual conditions of radiation therapy in humans, a rare effort in in-vitro research studies according to the researchers. Irradiation was performed in daily sessions with the same radiation therapy machine used in cancer patients, with flasks of cell cultures within an acrylic water platform, which mimics a human body with tumors. “Our objective was to duplicate the clinical situation as nearly as possible,” says Andrade.

The results of the combined therapies were quite encouraging. The researchers observed a reduction in treated cells of the levels of two proteins, ADAM17 and EFGR, associated with radiation resistance. More importantly, there was a decrease of more than 90% in the formation of new tumor cell colonies after the combined treatment. Compared to the control group, radiation therapy alone reduced colony formation by 36%. Even with the use of about half the standard dose of radiation, the probability of survival of the tumor remained low. This indicates that the combined use of radiation therapy and proteins that block calcium in the nucleus of cancer cells would fight the tumor more efficiently, exposing the patient to less damage than standard treatment.

“It’s a very elegant proof of concept,” says Roger Chammas, an oncologist at the University of São Paulo. He further points out that although the study used cancer cells of the head and neck, the proposed therapy, in principle, could be applied to other types of cancers that are resistant to radiation therapy. Helena Segretto, an oncologist and radiation biology expert at the Federal University of São Paulo (Unifesp), notes that radiation therapy combined with calcium blocking paralyzed the process of tumor cell division during the phases when these cells are more sensitive to radiation, inducing an increase in cell death. “Improving the response of tumor cells to radiation is the greatest desire of medical radiation therapists,” she says.

All the experts, however, agree that there is still a long way to go to prove the viability of this strategy. Segretto says that before the first tests in humans, experiments with laboratory animals will be required in which tumor cells interact with healthy ones, to be sure that they are unaffected by the therapy. “The first animal tests have begun and partnerships for future trials with teams from the Hospital das Clínicas of UFMG are already underway,” says Leite.

Scientific articles
ANDRADE, L.M. et al. Nucleoplasmic calcium buffering sensitizes human squamous cell carcinoma to anticancer therapy. Journal of Cancer Science and Therapy, May 25, 2012.
RODRIGUES, M.A. et al. Nucleoplasmic calcium is required for cell proliferation. Journal of Biological Chemistry, v. 282, June 8, 2007.