**1. Introduction**

Colorectal cancer (CRC) is the third most common and the second leading cause of death by cancer worldwide [1]. As CRC patients die of metastatic disease, prevention of the development of metastasis is essential to improve cure rates.

To metastasize, a tumor cell has to invade the surrounding tissue, enter the bloodstream, survive in circulation, and extravasate and colonize the distant organ, a process that requires multiple interactions between the malignant cell and its microenvironment [2].

**Citation:** Silva, S.; Cavaco, A.; Basso, B.; Mota, J.; Cruz-Duarte, R.; Costa, M.; Carvalho, L.; Lima, A.; Costa, L.; Ferreira, R.; et al. Therapeutic Potential of Deflamin against Colorectal Cancer Development and Progression. *Cancers* **2022**, *14*, 6182. https://doi.org/10.3390/ cancers14246182

Academic Editors: Barbara De Filippis, Alessandra Ammazzalorso and Marialuigia Fantacuzzi

Received: 8 November 2022 Accepted: 7 December 2022 Published: 14 December 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Matrix metalloproteinases (MMPs) comprise a large family of homologous zinc-dependent endoproteases, several of which play critical roles in this process, not only by degrading the extracellular matrix (ECM), allowing tumor dissemination and seeding but also by promoting angiogenesis and inflammation [3]. Therefore, increased MMP expression was detected in malignant tissues and was correlated with metastatic spread and unfavorable prognosis in multiple types of cancer [4,5]. Expectedly, MMPs were seen as ideal pharmacological targets for cancer therapy with preclinical studies of synthetic MMP inhibitors (MMPIs) holding great promise [6,7]. However, clinical trials developed during the late 1990s and early 2000s were unsuccessful in showing MMPIs effect in reducing tumor burden or improving overall survival, in addition to demonstrating severe side effects [8–10]. Further studies led to the conclusion that some MMPs, such as MMP-8, have anti-tumor effects, therefore the broad-spectrum of MMPIs was shown to be counter-productive, ultimately resulting in tumor progression and overall intense toxicity [11]. Thereby, it has now become apparent that to successfully target MMPs in the setting of cancer therapy, in situ inhibition of specific MMPs is essential. In particular, inhibition of the gelatinases MMP-2 and MMP-9 is envisaged, given their involvement in the degradation of the ECM and basement membrane, but also due to their role in the proteolysis of cell adhesion molecules and other bioactive proteins. In this context, old drugs and common natural products are now being explored for their potential to inhibit MMP-2 and MMP-9. Importantly, compounds derived from foods have been showing encouraging results in this regard, for example, curcumin, a component of the South Asian spice turmeric, has been shown to decrease MMP-2, -9, and -14 expression in various cancers, leading to decreased MMP activity and decreased cancer cell migration and invasion [12–14]. Similarly, antioxidant polyphenols in common foods, such as trans-resveratrol and quercetin from grapes and wine, as well as oleuropein and hydroxytyrosol from olive oil have also been shown to decrease the expression and activity of MMP-9 and decrease cancer cell migration, invasion, and angiogenesis [15–17]. Experimental studies of animal models fed with legume seeds have shown to reduce both the incidence and the number of colon tumors by 50% [18] and clinical studies have now started to provide evidence that legume consumption can decrease the risk of CRC [19]. In agreement with these reports, the World Cancer Research Fund/American Institute for Cancer Research recognized the potential of legume consumption in CRC prevention, supporting the need for additional research in this area.

We have recently discovered deflamin (patent WO/2018/060528), an oligomeric polypeptide isolated from the edible seeds of white lupin (*Lupinus albus*), that reduces MMP-2 and MMP-9 activity in CRC cell lines, in a dose-dependent manner and with an IC50 of 10 μg/mL. Being of food origin, and an oligomer of two storage proteins in legume seeds, deflamin isolated from white lupine was found to be safe for consumption, without impairing gene expression nor exerting cytotoxicity [20–23]. Its gelatinase inhibitory features suggested that it can be used in pathologies related to enhanced gastrointestinal MMP-9 activity, namely cancer, and inflammatory diseases. So far, deflamin was only successfully tested as a nutraceutical in inflammatory bowel disease models. In mice models of colitis, it significantly inhibited colonic MMP-9 activities, whilst reducing inflammation and colitis-induced lesions, when administered orally as a lupin seed extract or as a food additive to wheat cookies [21,24]. These works showed that deflamin is not only an efficient MMP-9 and MMP-2 inhibitor, but it also is highly resistant to digestion and not absorbed by the digestive tract (our unpublished results). These features infer that deflamin holds the potential to act locally in the intestinal path, bypassing the problem of systemic toxicity associated with common MMPIs [20–24]. Moreover, since it is water-soluble and rather effortlessly isolated [20], deflamin can easily become a novel nutraceutical for pathologies related to aberrant MMP-9 activities, particularly in the digestive system, such as CRC [22]. However, despite its potential against this disease, deflamin has never been tested in more realistic cancer models. In this context, we sought to explore the nutraceutical anti-tumor potential of this polypeptide in CRC progression. Not only this is the first report of deflamin in CRC models, but it also opens a door to novel approaches to tackle this disease.

### **2. Materials and Methods**

#### *2.1. Cell Lines*

The following colorectal carcinoma cell lines were used in this work: HT-29 (ECACC, no. 91072201), HCT116 (ATCC® CCL-247), and SW480 (ATCC® CCL-228). All cell lines were cultivated in DMEM media supplemented with 10% (*v*/*v*) FBS and 1% penicillin/streptomycin at 37 ◦C and 5% CO2, in a humidified atmosphere. Cells were maintained at a low passage and routinely tested for mycoplasma contamination by qPCR. Cell lines were validated by short tandem repeat (STR) profile.
