Protein extracted from plant inhibits the growth and migration of tumors

A molecule isolated from Enterolobium contortisiliquum (Pacara Earpod Tree) seeds shows anti-inflammatory, anticoagulant and antithrombotic action in in vitro tests and in animals.

By Karina Toledo, Caxambu

Agência FAPESP – A protein extracted from the seeds of Enterolobium contortisiliquum, popularly known as the Pacara Earpod Tree or monkey ear tree, shows potent anti-tumor, anti-inflammatory, anti-coagulant and anti-thrombotic properties in pre-clinical tests.

The in vitro and animal tests were conducted under the auspices of a thematic project funded by FAPESP and coordinated by Maria Luiza Vilela Oliva, a professor at the Universidade Federal de São Paulo (Unifesp). The results were presented during the 28th Meeting of the Federation of Experimental Biology Societies (FeSBE), which was held in Caxambu (MG) between August 21 and 24.

Oliva isolated the protein, named EcTI (Enterolobium contortisiliquum trypsin inhibitor), during her doctorate work at the end of the 1980s.

“We are seeking molecules capable of inhibiting the action of proteases – enzymes that break the peptide connections of other proteins. Such molecules are involved in numerous physiological and pathological processes in organisms, and an inhibitor can have interesting therapeutic effects,” explained Oliva.

The anti-tumor action was identified at the beginning of 2000 during another research project, and the molecule was patented in 2004.

“We called the molecule EcTI, a trypsin inhibitor, as this was the model enzyme we initially studied because it was cheaper. However, EcTI is patented as a protease inhibitor. We also patented its anti-cancer action,” stated Oliva.

The anti-tumor effect of EcTI was tested in cellular lines of breast, prostate, colorectal, blood (leukemia) and skin (melanoma) cancers, and the molecule inhibited in vitro cellular proliferation in all models. A portion of the results was reported in an article published in Biological Chemistry.

The researchers verified that EcTI impeded the adhesion of a line of gastric cancer cells to conjunctive tissue and, consequently, blocked invasion and cellular migration. These results were published in The Journal of Biological Chemistry.

“Before migrating to other tissues, the tumor cell must adhere to the conjunctive tissues that support it. EcTI blocks the protease found in the extracellular matrix and the signaling pathways used by the tumor in this process without affecting fibroblasts, which are healthy cells of conjunctive tissues,” explained Oliva.

Using a melanoma cell line, the researchers tested a treatment that associated EcTI with the chemotherapy drug 5-fluoracil. “The chemotherapeutic dose required to kill a cancer cell was 100 times lower when associated with EcTI. If this effect is proven in vivo, it will represent an enormous reduction in the side effects of chemotherapy,” said the researcher.

In tests conducted with mice, the researchers induced the development of melanoma and, in parallel, treated the animals with subcutaneous injections of EcTI for 20 days.

The control group that received only a placebo developed tumors, whereas tumor growth was inhibited by at least 90% in the group treated with EcTI. “Some animals never even developed the tumor,“ stated Oliva.

The researcher added that EcTI proved to be capable of impeding pulmonary metastasis in the same animal melanoma model.

Antithrombotic properties

In tests with rodents, the researchers observed that EcTI blocked the thrombosis process – a common complication among cancer patients subjected to chemotherapy.

“This protein inhibits the action of kallikrein, an enzyme that initiates the process of blood coagulation and also platelet aggregation. The effect was verified both in a rat model of arterial thrombosis and a mouse model of venomous thrombosis,” said Oliva.

In a project coordinated by researcher Iolanda de Fátima Lopes Calvo Tibério, of the Universidade de São Paulo Medical School (FMUSP), the action of EcTI on pulmonary inflammation has been tested with encouraging results in animal models of asthma and chronic obstructive pulmonary disease.

“We are now studying several peptides derived from this protein to determine whether they maintain the same protease inhibition action and whether there are other unknown effects. In in vitro tests, one of the peptides showed the capacity to impede the Trypanosoma cruzi parasite, which causes Chagas disease, from invading host cells. We intend to test the effect in animals,” commented the Unifesp professor.

According to the researcher, to date, EcTI has not presented any toxic effects in rodents receiving doses of 4 milligrams per kilogram. Nonetheless, more in-depth tests must still be conducted before testing the protein in humans.

“Large quantities of the protein and money are required to perform toxicity studies. A clinical trial will only be possible through a partnership with the pharmaceutical industry,” said Oliva.

Until this occurs, the group is studying several strategies for large-scale EcTI acquisition and the administration of the molecules in guinea pigs. “We need to evaluate, for example, whether it is more advantageous to cultivate the plant and isolate the protein or to produce it in a recombinant form (produced in bacteria or other microorganisms) in the laboratory. We also forming a partnership for the possibility of inserting the protein into nanocapsules and administering orally or via gavage (through the esophagus),” she commented.

Other inhibitors

Two other protease inhibitors, in the plant Bauhinia bauhinioides, were identified by the Oliva group during the FAPESP thematic project.

“These are two different proteins extracted from the same plant and named BbCI (Bauhinia bauhinoides cruzipain inhibitor) and BbKI (Bauhinia bauhinioides kallikrein inhibitor). The two proteins act through different pathways, and we are proving their therapeutic effects using animal cancer, thrombosis, inflammation and diabetes models.”