Published OnlineFirst February 26, 2018; DOI: 10.1158/1535-7163.MCT-17-0193 Small Molecule Therapeutics Molecular Cancer Therapeutics A Tricin Derivative from Deschampsia antarctica Desv. Inhibits Colorectal Carcinoma Growth and Liver Metastasis through the Induction of a Specific Immune Response Mariana Malvicini1, Ana Gutierrez-Moraga2,3, Marcelo M. Rodriguez1,Sofia Gomez-Bustillo1, Lorena Salazar2, Carlos Sunkel2, Leonor Nozal4, Antonio Salgado4, Manuel Hidalgo5, Pedro P. Lopez-Casas6, Jose Luis Novella4, Juan Jose Vaquero4, Julio Alvarez-Builla4, Adda Mora7, Manuel Gidekel2,7,8, and Guillermo Mazzolini1 Abstract In colorectal carcinoma patients, distant metastatic disease is by tumor rechallenge experiments. Antartina inhibits in vitro present at initial diagnosis in nearly 25% of them. The majority of human colorectal carcinoma cell proliferation; however, in vivo patients with metastatic colorectal carcinoma have incurable dis- experiments in Avatar colorectal carcinoma model Antartina dis- ease; therefore, new therapies are needed. Agents derived from play a limited antitumor effect. In an immunocompetent colorec- medicinal plants have already demonstrated therapeutic activities tal carcinoma mice model, Antartina potently inhibited tumor in human cancer cells. Antartina is an antitumor agent isolated growth and liver metastases, leading to complete tumor regressions from Deschampsia antarctica Desv. This study aimed to evaluate the in >30% of mice and increased animal survival. In addition, antitumor properties of Antartina in colorectal carcinoma models. Antartina induced a potent specific cytotoxic T-cell response We used human and murine colorectal carcinoma cell lines for against colorectal carcinoma and a long-lasting antitumor immu- investigating proliferation, apoptosis, and cell-cycle effects of nity. Interestingly, Antartina increased tumor immunogenicity and Antartina therapy in vitro. Avatar and immunocompetent colorec- stimulated dendritic cell activation. No toxic effects were observed tal carcinoma animal models were applied for evaluating the at the doses employed. Our findings showed that Antartina has the effects of Antartina in vivo. Immune response against colorectal ability to induce antitumor immunity against colorectal carcinoma carcinoma model was investigated using CTL assay, analyzing andcan be used to develop new tools for the treatment of colorectal dendritic cell activation and intratumor T-cell subpopulation, and carcinoma. Mol Cancer Ther; 17(5); 966–76. Ó2018 AACR. Introduction a potential curative option, but less than 20% to 30% of patients are suitable for resection due to clinical or technical causes, Colorectal carcinoma is a major cause of cancer-related mor- including extrahepatic disease (2). In the past few years, multi- bidity and mortality worldwide and is responsible for nearly disciplinary approaches such as chemotherapy regimens, radio- 700,000 deaths each year (1). For metastatic disease, surgery is embolization, and targeted therapies have been applied to improve patient survival, but as noted above still remains signif- icant mortality (3). Therefore, new therapeutic therapies are 1Gene Therapy Laboratory, Instituto de Investigaciones en Medicina Traslacional, needed for patients with a more advanced stage of the disease (4). Facultad de Ciencias Biomedicas, CONICET- Universidad Austral, Buenos Aires, Argentina. 2Universidad Autonoma de Chile, Providencia, Chile. 3Universidad de With its unique weather and environmental characteristics, La Frontera, Temuco, Chile. 4Departamento de Química Organica, Universidad Antarctica is home to an extraordinary variety of extremophile de Alcala, Madrid, Spain. 5Beth Israel Deaconess Medical Center, Harvard Medical organisms, including vascular plants such as Deschampsia School, Boston, Massachusetts. 6Gastrointestinal Cancer Clinical Research Unit, antarctica (5). There are several examples of novel psychrophilic 7 CNIO - Spanish National Cancer Research Center, Madrid, Spain. Creative enzymes and new molecules isolated from organisms found in 8 BioScience, Huechuraba, Santiago, Chile. Department of Entomology & Nem- Antarctica with potential uses in medicine (6). In particular, atology, University of California, Davis, Davis, California. Deschampsia antarctica is able to tolerate high UV exposure due Note: Supplementary data for this article are available at Molecular Cancer to the production of secondary metabolites as photoprotector Therapeutics Online (http://mct.aacrjournals.org/). agents, especially flavonoid-like molecules (7, 8). Tricin, a flavo- M. Gidekel and G. Mazzolini are co-senior authors of this article. noid type compound from the secondary metabolic pathways in Corresponding Authors: Guillermo Mazzolini, CONICET-Universidad Austral, plants, has importance to plant growth by protecting against Av. Presidente Peron 1500, Pilar, Buenos Aires B1629ODT, Argentina. Phone: disease, weeds, and microorganisms (9). Tricin is present in 5423-2048-42618; Fax: 5423-2248-2205; E-mail: [email protected]; herbaceous and cereal plants and exists as free tricin and its and Manuel Gidekel, Universidad Autonoma de Chile, Providencia, Chile. E-mail: derivatives, such as tricin-glycosides (9). The flavone tricin and [email protected] their derivatives have been shown to exhibit antiproliferative doi: 10.1158/1535-7163.MCT-17-0193 activity against several human cancer cell lines, cancer chemo- Ó2018 American Association for Cancer Research. protective effects in the gastrointestinal tract of mice, and 966 Mol Cancer Ther; 17(5) May 2018 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst February 26, 2018; DOI: 10.1158/1535-7163.MCT-17-0193 Antartina Induces Immunity against Colorectal Carcinoma antioxidative effects (10–13). The reported antitumor activities of 37 C in a 5% CO2 humidified atmosphere. For in vitro assays, cells flavonoids involve the inhibition of proliferation and induction were thawed and cultured generally for 48 hours, until 90% to of apoptosis, suppression of protein tyrosine kinase activity, and 100% confluence. Then, cells were dissociated using tripsin, antiangiogenic effects (14). However, little is known about the passed, and cultured for another 48 hours. For in vivo experiments, role of tricin derivatives and its bioactivity as anticancer molecule. cells were cultured at least for one week with two or three passages Antartina (tricin 7-O-beta-D glucopyranoside) was isolated from until inoculation. the Antarctic plant Deschampsia antarctica and has been previously reported from other plants (15, 16). Because of low natural Drugs availability, a synthesis was conducted, and herein, we report on Antartina was dissolved in 0.1% DMSO and sterile water and the antitumor activity of Antartina produced synthetically. injected intraperitoneally at the doses and the schedule indicated In this work, we show that Antartina induces a potent and or used for in vitro assays at different concentrations (0.1, 1, 10, specific immune response against colorectal carcinoma in mice. 100, and 1,000 mg/mL). Tricin was dissolved in 0.3% DMSO Therefore, Antartina represents a promising therapeutic agent for and sterile water and injected intraperitoneally. Fluorouracil colorectal carcinoma patients. Rontag (5-FU) 500 mg was diluted in saline at indicated con- centrations for in vitro assays or injected intraperitoneally for Materials and Methods in vivo experiments. Antartina In vitro experiments Tricin 7-O-b-D-glucopyranoside (initially called peak 10; molecular formula: C H O ; CAS no. 32769-01-0, purity Cell viability assays. LoVo, ASG, Wi38, and CT26 cells were plated 23 24 12  3 >99.2%) was detected and isolated from aqueous extracts of onto 96-well plates at a density of 5 10 cells per well and Deschampsia antarctica Desv.(Poaceae). Synthetic Antartina was cultured with DMEM 0.1% DMSO or Antartina at different doses. supplied by the Centro de Química Aplicada y Biotecnología Forty-eight and 72 hours after treatment, viability was determined (CQAB), Universidad de Alcala, Alcala de Henares (Madrid, by the MTS assay (Promega). The plates were incubated for 4 Spain). To determine the structure of Antartina, a yellow amor- hours; the absorbance of each well was read at 490 nm. All assays phous solid, encoded as peak 10, was used, obtained from were performed in quadruplicate, and each assay was repeated at aqueous extract MCC12-034-03 (JON 2009) according to meth- least twice. Morphologic features associated with apoptosis were odology developed by CQAB, by solid phase extraction and then analyzed by acridine orange (AO) and ethidium bromide (EB) m semipreparative high-performance liquid chromatography staining. CT26 cells were treated with 10 g/mL Antartina, and (HPLC). The mass spectrum (MS) analysis using the internal 24 or 72 hours later, cells were resuspended in the dye mixture m m CQAB method confirms that the purity of the isolated sample (100 g/mL AO and 100 g/mL EB in PBS) and visualized by fl was greater than 99.2% (detection at 254 nm). The MS, in uorescence microscopy (Nikon Eclipse E800). þ electrospray positive mode (ESI ), gives rise to a peak with a  6 mass/charge ratio (m/z 493), whereas in electrospray negative Cell-cycle analysis. CT26 cells (1 10 ) cultured with DMEM or À fi mode (ESI ) an m/z signal 491. This indicates a molecular mass Antartina were collected, washed in PBS, and xed in a mixture of for the product