Use of beta-conglutin proteins as radiosensitising agents

Abstract

The present invention relates to β-conglutin proteins of the blue lupin seed (Lupinus angustifolius L.), preferably β1, β3 and β6 isoforms, for use thereof as an adjuvant and radiosensitiser in the treatment of cancer, preferably breast cancer and more preferably, triple-negative breast cancer, with radiotherapy with ionising radiation.

Description

[0001] USE OF BETA CONGLUTIN PROTEINS AS RADIOSENSITIZING AGENTS

[0002] DESCRIPTION

[0003] TECHNICAL FIELD

[0004] The present invention falls within the field of medicine, specifically within therapies for the treatment of cancer, and aims to identify new radiosensitizing agents that can increase the sensitivity of tumor cells to radiotherapy.

[0005] STATE OF THE PRIOR ART

[0006] Breast cancer (BC) is the most prevalent tumor among women, the second most common worldwide, and the fifth in terms of mortality. Clinically, BC is a heterogeneous disease with significant differences in prognosis, treatment, and success rates. At the molecular level, BC is classified based on the presence or absence of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2). It is categorized as Luminal A (ER+ and PR+, HER2-), with a better prognosis, slow growth rate, and lower aggressive potential; Luminal B (ER+ and PR+, HER2+), with a worse prognosis and faster growth rate; or Triple negative (ER-, PR-, and HER2-), which is found in women with BRCA1 mutations and corresponds to infiltrating ductal tumors with high rates of metastasis.

[0007] Currently, the main treatments for BC include surgery, hormone therapy, chemotherapy, radiation therapy (RT), and immunotherapy. Therapies may have limited efficacy depending on the molecular type, making BC extremely difficult to treat.

[0008] Ionizing radiation (IR) in RT is used to treat most tumors, as RI damages cells. RI activates the antitumor immune response, activating signaling pathways that are normally suppressed by the tumor, such as cell death or immune suppressor cells.

[0009] RT is a highly selective and effective treatment for a large number of tumors. Its success in tumor eradication depends mainly on the total radiation dose administered, which in turn is limited by the tolerance of the normal tissue surrounding the tumor. Specifically, in BC, the most commonly applied radiotherapy regimen is conventional RT (daily fractions of 2 Gy), achieving a total dose of 45–50 Gy. However, in recent decades, hypofractionated RT, which involves higher radiation doses (fractions >2 Gy) in shorter treatment times, has become more common. Hypofractionated RT delivers doses ranging from 2.66 Gy to more than 6 Gy per session, which allows for shorter treatment times while maintaining and even increasing the efficacy of conventional RT.

[0010] Another limitation of RT is related to the intrinsic radioresistance of the tumor, due to the presence of cancer stem cells (CSCs) that can survive conventional RT regimens and repopulate the tumor, contributing to tumor recurrence and metastasis. It has been shown that low doses of radiation enhance the enrichment of the cellular fraction with a higher capacity for self-renewal that expresses CSC markers, so one of the major limitations of RT is the collateral induction of metastasis, the CSC phenotype and oncogenic metabolism [1],

[0011] CSCs have certain characteristics of normal stem / progenitor cells but a greater tumorigenic potential. Furthermore, they have a high capacity to repair DNA damage, unlimited self-renewal, proliferation, and differentiation into other cell lines, and high survival rates in the bloodstream. Therefore, CSCs play an important role in cancer treatment resistance and recurrence or relapse, making CSC-targeted therapies key to complete tumor eradication.

[0012] These reasons explain the emergence of new radiosensitizers as adjuvants to enhance the effects of RT that allow systemic control of the disease and improve oncological results [2]. Some nutraceutical compounds of plant origin have shown promising results in this regard.

[0013] Legume seed proteins have demonstrated health benefits as functional foods. In particular, the seeds of Lupinus angustifolius L., blue lupin or narrow-leaf lupin, have shown their potential for improvement in inflammatory-based diseases such as type 2 diabetes. Recent studies [3-6] have shown that one of the main compounds in these seeds, conglutin p proteins, mediates the improvement of the effects of inflammation-related diseases at the molecular level, due to its multifunctionality [3], Conglutin proteins belong to the family of proteins called vicilins or 7S globulins, and 7 functional genes (pi to 7) are identified in them [7], some of which provide their antioxidant, anti-inflammatory and antidiabetic capacity [3],

[0014] The amino acid sequences of the pi-7 conglutin proteins isolated from blue lupin seed and the accession number of the coding mRNA nucleotide sequence in the GenBank database (https: / / www.ncbi.nlm.nih.gov / nuccore / ) for each of them are provided below.

[0015] The amino acid sequence of the conglutin I protein isolated from blue lupin seed; SEQ ID NO: 1, with accession number F5B8V9 within the protein database called Uniprot (https: / / www.uniprot.org / ), is as follows:

[0016] MAKMRVRLPMLILLLGWFLASIGIAYGEKDFTKNPPKEREEEEHEPRQQPRPRQQEE QEREHRREEKHDGEPSRGRSQSEESQEEEHERRREHHREREQEQPRPQRRQEEEE EEEEWQPRRQRPQSRREEREREQEQGSSSGSQRGDERRQRRQHRREQHRREE EQDSRSDSRRQRNPYHFSSNRFQTYYRNRNGQIRVLERFNQRTNRLENLQNYRIIEFQS KPNTLILPKHSDADFILVLNGRATITIVNPDKRQVYNLEQGDALRLPAGTSYILNPDDNQ NLRVAKLAIPINNPPGKLYDFYPSTTKDQQSYFSGFSKNTLEATFNTRYEEIERVLLGDDEL QENEKQRRGQEQSHQDEGVIVRVSKKQQIQELRKHAQSSSGEGKPSESGPPFNLRNSNKPI YSNKFGNFYEITPDINPQFQDLNISLTFELFGENCEWGIVDEGIVDE RDQQRQQDEQEEEQGEEEVRRYSDKLSKGDVFIIPAGHPLSINASSNLRLLGFGINAN ENQRNFLAGSEDNVIKQLDREVKELTFPGSIEDVERLIKNQQQSYFANAQPQQQQQREK EGRRGRRGPISSILNALY

[0017] Similarly, conglutin protein i can be identified by the nucleotide sequence of coding mRNA; with accession number HQ670409 in GenBank, SEQ ID NO: 8 ( Figure 2 ).

[0018] The amino acid sequence of conglutin 2 protein isolated from blue altramuz seed; SEQ ID NO: 2, with accession number F5B8W0 in Uniprot, is as follows

[0019] MANMRVKFPTLVLLLGIVFLMAVSIGIAYGEKNAIKNHERPQEREQEERDPRQQPRPRHQ EEQEREHGREEERNREPSRGRSESEESREEEREQRREPSRGREQEQQPQHGRREEE EEWQPRRQRPQSRREEREQEQGSSSSSGRQSGYERREQREEREQQQEQDSRSESR RQRNPYYFSYERFQTLYKNRNGQIRVLERFDQRTNRLENLQNYRIVEFQSKPNTLILPKH SDADYILVVLNGRATIVNPDKRQAYNLEHGDALRLPAGTTSYILNPDDNQNLRWKLAIP INNPGNFYDFYPSSTKDQQSYFNGFSRNTLEATFNTRYEEIQRIILGNEDGQEDEEQSRG QEQSHQDQGVIVRVSKEQIQELRKHAQSSSGKGKPSESGPFNLRSDEPIYSNKFGNFYE ITPDRNPQAQDLDISLTFIEINEGGLLLPHYNSKAIFVWVDEGEGNYELVGIRDQERQQD EQEQEEVRRYNAKLSEGDIFVIPAGHPISINASSNLRLLGFGINADENQRNFLAGSEDNVI RQLDKEVKQLTFPGSVEDVERLIKNQQQSYFANAQPQQQQREKEGRRGRRGLSFPFR SLFTKLLSTIM

[0020] Similarly, conglutin protein p2 can be identified by the nucleotide sequence of coding mRNA; with accession number HQ670410 in GenBank, SEQ ID NO: 9 (Figure 2).<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0021] <h2 style=";text-align:left;direction:ltr"> The aminoacídica secuencia of the protein conglutina p3 aislada of semilla of ultramuz aul; SEQ ID NO: 3, with the F5B8W1 attached to the Uniprot, with the following:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0022] <h2 style=";text-align:left;direction:ltr"> MAKMRVRFPTLVLLLGIVFLMAVSIGIAYGEKNVLKNHERPQEREQEERDPRQQPRPHH QEEQEREHRRESEESQEEEREQRREPRREREQEQQPQHGRREEEEEWQPRRQRPQS RREEREQEQGSSSSSRRQSGYERREQREEREQEQEQEQGSRSDSRRQRNPYYFSSERF QTLYRNRNGQIRVLERFDQRTNRLENLQNYRIVEFQSKPNTLILPKHSDADYILVVLNGSA TITIVNPDKRQSYNLENGDALRLPAGTTSYILNPDDNQNLRVVKLAIPINNPGNFYDFYPSS SKDQQSYFSGFSKNTLEATFNTRYEEIQSILLGNEDEQEDDEQWHGQEQSHQDEGVIVR VSKEQVQELRKYAQSSSRKGKPYESGPFNLRSNKPIYSNKFGNFYEITPDRNPQAQDLDI SLTFIEINEGALLLPHYNSKAIFVWVDEGEGNYELVGIRDQQRQQDEQEVRRYSARLSE GDIFVIPAGHPISINASSNLRLLGFGINADENQRNFLAGSEDNVIRQLDREVKGLIFPGSAE DVERLIKNQQQSYFANAQPQQQQQREREGRHGRRGHISSILSTLY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0023] Similarly, p3 conglutin protein can be identified by the nucleotide sequence of coding mRNA; with accession number HQ670411 in GenBank, SEQ ID NO: 10 (Figure 2).

[0024] The amino acid sequence of conglutin p4 protein isolated from blue altramuz seed; SEQ ID NO: 4, accession number F5B8W2 in Uniprot, is as follows:

[0025] MIKMRVRFPTLVLLLGIVFLMAVSIGIAYGEKNVIKNHERPQEREQEERDPRQQPRPHHQ EEQEREHRREEERDREPSRGRSESEESREEEREQRREPRREREQEQQPQHGRREEE EEWQPRRQRPQSRREEREQEQGSSSSSRRQSGYERREEREQEQEQGSRSDSRRQR NPYYFSSERFQTLYRNRNGQIRVLERFDQRTDRLENLQNYRIVEFQSKPNTLILPKHSDA DYILWLNGSATITIVNPDKRQSYNLENGDALRLPAGTTSYILNPDDNQNLRVVKLAIPINN PGNFYDFYPSSSKDQQSYFSGFSRNTLEATFNTRYEEIQRILLGNEDEQEDDEQRHGQE QSHQDEGVIVRVSKEQVQELRKYAQSSSRKGKPSKSGPFNLRSNKPIYSNKFGNFYEIT PNRNPQAQDLDISLTFIEINEGALLLPHYNSKAIFVVLVDEGEGNYELVGIRDQQRQQDEQ EVRRYSARLSEGDIFVIPAGHPISINASSNLRLLGFGINADENQRNFLAGSEDNVIRQLDTE

[0026] VKGLTFPGSTEDVERLIKNQQQSYFANAQPQQQQQREREGRRGRRGHISSILSTLY

[0027] Similarly, conglutin p4 protein can be identified by the nucleotide sequence of the coding mRNA; with accession number HQ670412 in GenBank, SEQ ID NO: 11 (Figure 2).

[0028] The amino acid sequence of the conglutin p5 protein isolated from blue lupin seed; SEQ ID NO: 5, Uniprot accession number F5B8W3, is as follows:

[0029] MAKMRVRFPMLVLLLGFLAVSIGIAYGEKDVIKNPERPEERQEEERDPRQPPRSRQ QEEQEREHRREKERDREPSRGRSESKQSQEEERERKHDREREQQPQYGRRHE EEEKGEEEEEGQARRQRPQRRREQEQGSSRESQRRREQREQREQREQREQREQPQPPRSRQ EQEQGSSSGRQSDYGRRQRHEGREQEQGSSSESHRLRNPYFSSERFQTR YKNKNGQIRVLERFDNRLENLQNYRIVEFQSRPNTLILPKHSDADYILVVLNGRATITI VNPDKRQAYNLEYGDALRPAGTTSYILNPQLAQDKVKDKYFKYFGRTK DQQSYFSGFSKNTLEATFNTHYEEIQRILLGYEDEQEDEEQRRREQEQSHQDEGVIVRVS KEQIQELRKHAQSSSRKGKPSESGPPFNLRSNEPIYSNKFGNFYEITPDRNPQVQDLDISLI FTEISEGALLLPHYNSKAIFVIVVDEGEGNYELVGIRNQQRQQDEQEVEEVRSYNARLSE GDILVIPAGHPLSINASSNLRLLGFGINADENQRNFLAGSEDNVIRQLDREVKELIFPGSAE DVERLIRNQQQSYFANAQPQQQRRQQREKREKLRGRPSINARLSE

[0030] Similarly, conglutin protein p5 can be identified by the nucleotide sequence of coding mRNA; with accession number HQ670413 in GenBank, SEQ ID NO: 12 ( Figure 2 ).

[0031] The amino acid sequence of conglutin p6 protein isolated from blue altramuz seed; SEQ ID NO: 6, accession number F5B8W4 in Uniprot, is as follows:

[0032] MIKMRVRFPTLVLLLGIVFLMAVSIGIAYGEKNVIKNHERPQEREQEERDPRQQPRPHHQ EEQEREHRREEERDREPSRGRRESEESREEEREQRREPRREREQEQQPQHGRREEE EEWQPRRQRPQSRREEREQEQGSSSSSRRQSAYERREQREEREQEQGGSRSDSRR QRNPYYFSSERFQTLYRNRNGQIRVLERFDKRTDRLENLQNYRIVEFQSKPNTLILPKHS DADYILWLNGSATITIVNPDKRQSYNLENGDALRLPAGTTSYILNPDDNQNLRVVKLAIPI NNPGNFYDFYPSSSKDQQSYFSGFSRNTLEATFNTRYEEIQRILLGNEDEQEDDEQRHG QEQSHQDEGVIVRVSKEQVQELRKYAQSSSRKGKPSKSGPFNLRSNKPIYSNKFGNFYE ITPNRNPQAQDLDISLTFIEINEGALLLPHYNSKAIFVVLVDEGEGNYELVGIRDQQRQQDE QEVRRYSARLSEGDIFVIPAGHPISINASSNFRLLGFGINADENQRNFLAGFEDNVIRQLD REVKGLTFPGFAEDVERLIKNQQQSYFANAQPQQQQQREREGRHGRRGHIFSILSTLY Similarly, p6 conglutin protein can be identified by the nucleotide sequence of coding mRNA; with accession number HQ670414 in GenBank, SEQ ID NO: 13

[0033] (Figure 2).

[0034] The amino acid sequence of conglutin p7 protein isolated from blue alfalfa seed; SEQ ID NO: 7, with accession number F5B8W5 in Uniprot is as follows:

[0035] MARMRVRFPTLVLLLGILFLMAVSIGIAYGKDVIKNHERPGEREHEERDPRQQPRPRKQ EEQEREHRREEEHDRDPSRGRRESEERQEEERERRRREPCREREQQPQHGRREEE EEEEEWQPRRLRPQSRKEERQEQGSSSSSRQYERRQYERQEQEQEQEQEQQQQPRPRKQ SRSDSRRQRNPYHFSSERFQTRYRNRNGQIRVLERFDQRTNRLENLQNYRIVEFQSNP NTLILPKHSDADYILVVLNGRATITIVNPDKRQAYNLEYGDALRVPAGTTSYILNPDDNQNL RVVKLAIPINNPSNFYDFYPSSTKDQQSYFSGFSKNTLEATFNTRYEEIQRILLGNEDEQE DEEQRRGQQEQSYQDEGVIVRVSKEQIQELKHAQSSSRKGKPSESGPPFNLRSNESIYSN KFGNFYEITPERNPQVQDLDISLTFTEINEGALLLPHYNSKAIFIWVDEGEGNYELVGIRD QQRQQDEQEEEEEEEVRRYSARLSEGDIFVIPAGYPISVNASSNLRLLGFGINANENQRNF LAGEDNVISQLDREVKELTFPGSAQDVERLIKNQQQQQQQQQQQQQQQQQQQRYSARLGFGINANENQRNF LAGEDNVISQL RRSLISSILSTLY

[0036] Similarly, conglutin p7 protein can be identified by the nucleotide sequence of the coding mRNA; with accession number HQ670415 in GenBank, SEQ ID NO: 14 (Figure 2).

[0037] The structural features of conglutin p proteins are distinctive from other proteins of the vicilin family from legume species, since they include two structural domains that characterize the superfamily of proteins called cupin, which form a structure similar to the structural fold called "Rossmann" common in enzymes that use molecular oxygen as a substrate [8], All conglutin p proteins have two differentiated areas or domains: a globular domain and a mobile arm. A similar mobile arm has not been found in other vicilin proteins from legume species [9] so the conglutin p proteins of blue lupin are unique in terms of their structure, hence their potential and interest.

[0038] The properties of conglutin p proteins appear to be due to this mobile arm that they present at the N-terminal end, a structural domain rich in alpha helices. The mobile arm is the section that presents the greatest variability and without it none of the conglutin p proteins are functional. The analysis of molecular models has shown that the major structural differences between conglutin p isoforms are mainly found in this N-terminal mobile arm; therefore, the possible functional differences between conglutin p could be due to these structural differences [8],

[0039] The high homology between the 7 conglutin protein isoforms isolated from blue lupin can be seen in Figure 1.

[0040] The 7 proteins present a sequence identity percentage between 77.4 and 97.8% with respect to their amino acid sequences, as can be seen in Figure 2, and between 72.8% and 98.9% similarity with respect to their nucleotide sequences.

[0041] In particular, conglutins p1, p3, and p6 have been shown to exert a variety of effects on cells at different levels [3, 4, 6]: they reduce the mRNA expression of proinflammatory mediators, they reduce the chemotactic capacity of cells by decreasing the levels of chemokines and cell adhesion factors; they regulate cellular oxidative metabolism and they reverse insulin resistance [3, 4, 6], Due to these effects, conglutin p proteins from blue lupin seed could be used in the prevention and treatment of inflammatory-based diseases.

[0042] The cytotoxic and anti-potential properties of the conglutin proteins pi, p3 and p6 have been tested in an in vitro model of CM inducing a reduction in CM cell viability, higher apoptotic rates, reduction in self-renewal capacity, and in the number of CSCs through the regulation of the p53 / SIRT1 / FoxO-1 pathway, while preserving healthy cell lines

[0010] ,

[0043] BRIEF DESCRIPTION OF THE INVENTION

[0044] The present invention describes the radiosensitizing effect of blue lupin seed conglutin p proteins for use in combination with ionizing radiotherapy (IR) in the treatment of CM. Specifically, the radiosensitizing effect of the conglutin p protein isoforms, p3 and p6, is tested. This effect is presumably extensible to the rest of the conglutin p protein isoforms due to the high degree of structural and molecular homology that they present between them, as previously described.

[0045] The pi, p3, and p6 isoforms of blue lupin (Lupinus angustifolius L.) conglutinated p proteins were purified by affinity chromatography, and their potential as natural radiosensitizers for BC cells in vitro was evaluated with dose-response curves after colony formation assay. The survival fraction, apoptosis, DNA damage response, reactive oxygen species (ROS) production, and cell growth and self-renewal capacity as well as the underlying mechanisms of action after combined treatment with conglutinated p proteins and RT were assessed in three BC cell lines compared to a healthy cell line.

[0046] Conglutin proteins pi, 3 and p6, showed a remarkable radiosensitizing effect on the three BC cell lines analyzed, dependent on the molecular subtype, showing the greatest effects on the triple-negative phenotype cell line, MDA-MB-231. Conglutin proteins were effective at extremely low concentrations (2.5-10 ng / uL) when combined with standard doses of ionizing radiation (4-6 Gy) used in hypofractionated RT, highlighting their potential as natural sensitizers in combination with RT.

[0047] MCF-10A cells (healthy epithelial cells used as controls) pretreated with conglutin-β showed no significant changes in resistance to Rl at all doses tested, preserving the resistance of healthy cells. Therefore, purified conglutin-β proteins have the ability to selectively target CSCs while preserving the viability of healthy cells, which is crucial for effective cancer treatment.

[0048] The mechanism of action of conglutin p proteins appears to involve the modulation of tumorigenic genes related to cell growth, such as SIRT1 and FoxO1, depending on the p53 status and the dose of RI.

[0049] These nutraceutical proteins enhance the efficacy of RT while minimizing damage to healthy cells, making them a useful pretreatment strategy for patients with BC, reducing the risk of recurrence.

[0050] Therefore, a first aspect of the invention relates to the conglutin p protein isolated from seeds of Lupinus angustifolius L., hereinafter “protein of the invention”, for use as a radiosensitizer in radiotherapy.

[0051] Preferably, the protein of the invention has an identity of at least 95% or at least 99% or 100% with any of the amino acid sequences SEQ ID NO 1 to SEQ ID NO 7 and has the activity and structural characteristics of the respective conglutin proteins pi to p7. More preferably, the protein of the invention has an identity of at least 95% or at least 99% or 100% with SEQ ID NO 1 and has the activity and structural characteristics of the conglutin protein [31, or an identity of at least 95% or at least 99% or 100% with SEQ ID NO 3 and has the activity and structural characteristics of the conglutin protein |33, or an identity of at least 95% or at least 99% or 100% with SEQ ID NO 6 and has the activity and structural characteristics of the conglutin protein p6.

[0052] A second aspect of the invention relates to a composition comprising at least one protein of the invention for use as a radiosensitizer in radiotherapy, hereinafter “composition of the invention”.

[0053] Preferably, the composition comprises at least one protein that has an identity of at least 95% or at least 99% or 100% with any of the amino acid sequences SEQ ID NO 1 to SEQ ID NO 7 and has the activity and structural characteristics of the respective conglutin proteins pi to p7. More preferably, it comprises at least one protein that has an identity of at least 95% or at least 99% or 100% with SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 6 and that has the activity and structural characteristics of the conglutin proteins pi, p3, p6 respectively. More preferably, it comprises at least one protein of sequence SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO:6.

[0054] In a preferred embodiment of the composition of the invention, it is a pharmaceutical composition, and more preferably, it also comprises pharmaceutically acceptable excipients.

[0055] A third aspect of the invention describes the protein or composition of the invention for use as a radiosensitizer in a method using radiotherapy for the treatment of cancer. The cancer could be any type of cancer that is susceptible to the clinical benefit of radiotherapy treatment. Preferably, the type of cancer is selected from the list comprising colon cancer, pancreatic cancer, hepatocarcinoma, head and cervix cancer, bladder cancer, lung cancer, and breast cancer. More preferably, the cancer is breast cancer, and even more preferably, the breast cancer is triple-negative.

[0056] Throughout the description and claims, the word "comprise" and its variants are not intended to exclude other technical features, additives, components, or steps. For those skilled in the art, other objects, advantages, and features of the invention will be apparent in part from the description and in part from the practice of the invention. The following examples and drawings are provided for illustrative purposes only and are not intended to limit the present invention.

[0057] BRIEF DESCRIPTION OF THE FIGURES

[0058] Figure 1. 3D structures of conglutins pi to p7: α-helices (red), p-sheets (yellow) and spirals (green) are represented, integrating the main domains of the proteins.

[0059] Figure 2. Table with the alignments of the nucleotide sequences of conglutins P1 to P7 (SEQ ID NO 8 to SEQ ID NO 14).

[0060] Figure 3. Dose-response curves (survival fraction) for the four cell lines, (a) MCF10-A, (b) MCF-7, (c) SKBR-3 and (d) MDA-MB-231, under 24h treatment with selected concentrations of conglutin proteins pi, p3 and p6 and a combined added radiation dose of 0, 1, 2, 4, 6 and 8 Gy. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus untreated cells.

[0061] Figure 4: Apoptotic cells in an in vitro advanced BC model after 24 h of pretreatment with conglutin proteins pi (2.5 ng / pl), p3 (5 ng / pl), or p6 (10 ng / pl) and subsequent RI treatment at 4 and 6 Gy in (a) MCF-7, (b) SK-BR-3, and (c) MDA-MB-231. Cells were trypsinized 1 h after RI and incubated for 15 min with annexinV-FITC and propidium iodide-PI. The percentage of apoptosis was calculated using early and late apoptotic cells. Samples were analyzed using a BD FACS aria lllu flow cytometer. ** p < 0.01 and *** p < 0.001 versus untreated cells, and # p < 0.05, ## p < 0.01 and ### p < 0.001 versus other treatments.

[0062] Figure 5: Western blot analysis of total caspase-3. β-Actin was used as a control. Cells were treated with conglutin I (2.5 ng / μl), 3 (5 ng / μl), or 6 (10 ng / μl) proteins for 24 h in MCF-7, SK-BR-3, and MDA-MB-321 and then irradiated at 4 Gy. Protein was harvested 24 h after IR.

[0063] Figure 6: Western blot analysis of total caspase-3. p-Actin was used as a control. Cells were treated with conglutin proteins pi (2.5 ng / μl), p3 (5 ng / μl), or p6 (10 ng / μl) for 24 h in MCF-7, SK-BR-3, and MDA-MB-321 and then irradiated at 6 Gy. Protein was harvested 24 h after IR.

[0064] Figure 7: ROS levels after treatment with conglutin proteins pi (2.5 ng / pl), p3 (5 ng / pl), or P6 (10 ng / pl) for 24 h and subsequent Rl at 4 and 6 Gy in (a) MCF-7, (b) SK-BR-3, and (c) MDA-MB-321. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus untreated cells, and # p < 0.05, ## p < 0.01 versus other treatments. Figure 8: Phosphorylated yH2Ax (%) or DNA repair rate after treatment with conglutin proteins pi (2.5 ng / pl), p3 (5 ng / pl) or p6 (10 ng / pl) for 24h and subsequent Rl at 4 and 6 Gy in (a) MCF-7, (b) SK-BR-3 and (c) MDA-MB-321. * p < 0.05, ** p < 0.01 , and *** p < 0.001 vs. untreated cells, and # p < 0.05, ## p < 0.01 vs. other treatments.

[0065] Figure 9: Western blot analysis of SIRT1, FoxO1, LC3B and p62. p-actin was used as a control. Cells were treated with conglutin proteins pi (2.5 ng / pl), p3 (5 ng / pl) or P6 (10 ng / pl) for 24 h in MCF10-A, MCF-7, SK-BR-3 and MDA-MB-321 and irradiated at 4 Gy.

[0066] Figure 10: Western blot analysis of SIRT1, FoxO1, LC3B and p62. p-actin was used as a control. Cells were treated with conglutin proteins pi (2.5 ng / pl), p3 (5 ng / pl) or P6 (10 ng / pl) for 24 h in MCF10-A, MCF-7, SK-BR-3 and MDA-MB-321 and irradiated at 6 Gy.

[0067] Figure 11. ALDH1 activity following 24 h pretreatment with conglutin proteins P1 (2.5 ng / μl), p3 (5 ng / μl), or p6 (10 ng / μl) and subsequent Rl treatment at 4 and 6 Gy in (a) MCF-7, (b) SK-BR-3, and (c) MDA-MB-321. Viable ALDH1+ cells were quantified by flow cytometry 1 h post-Rl treatment on a BD FACS aha II flow cytometer lu * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. untreated cells and ## p < 0.01, ### p < 0.001 vs. other treatments.

[0068] Figure 12. CD44 / CD24highlow expression after 24h pretreatment with conglutin proteins pI (2.5 ng / pI), p3 (5 ng / pI), or p6 (10 ng / pI) and post-RI treatment at 4 Gy and 6 Gy in (a) MCF-7, (b) SK-BR-3, and (c) MDA-MB-321. Samples were analyzed by flow cytometry 1h post-RI on a BD FACS flow cytometer. ** p < 0.01, and *** p < 0.001 vs. untreated cells and # p < 0.05, ## p < 0.01 vs. other treatments.

[0069] Figure 13. Quantification of mammospheres after 24 h pretreatment with conglutin proteins pi (2.5 ng / pl), p3 (5 ng / pl), or p6 (10 ng / pl) and subsequent Rl treatment at 4 and 6 Gy. Rl treatment was performed 24 h before seeding mammospheres of (a) MCF-7, (b) SK-BR-3, and (c) MDAMB-231 and the spheres were characterized in (e). Spheres >50 pm were counted and captured using the binocular microscope at 10x magnification. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated cells.

[0070] DETAILED DESCRIPTION OF THE INVENTION Overexpression and purification of conglutin isoforms (3

[0071] To obtain the lupin conglutin p proteins, specifically the pi, 3 and 6 isoforms, the same protocol used in

[0010] was carried out, which includes overexpression and purification of these proteins based on a previous protocol

[0011] with modifications following the following steps:

[0072] - Construction of expression plasmids for each of the conglutin isoforms 1 3, 6 using a pET28b(+) vector (Novagen) with an N-terminal polyhistidine (6xHis) and an intermediate vector pUC57 containing a synthetic gene that codes for each conglutin protein.

[0073] - Overexpression of these conglutin protein isoforms using different expression induction methods (chemical and physical) in bacteria (Escherichia coli).

[0074] - Purification of conglutin p protein isoforms by combining biochemical techniques such as sonication of bacterial cells, differential centrifugation, tandem affinity chromatography with proteins bound to a His-tag, dialysis steps, following the protocol of Jiménez-López et al. 2016

[0011] with modifications.

[0075] - Identification of the p-conglutin proteins previously obtained by methods such as SDS-PAGE and immunoblotting using a highly specific anti-conglutin p antibody (Agrisera, Sweden) [4, 6, 11],

[0076] Cell culture

[0077] The cell lines used were obtained from ATCC, specifically, three BC tumor lines: MCF-7 (ERa positive, weak for HER2), SK-BR-3 (ERa negative, HER2 positive) and MDA-MB-231 (ERa negative, negative for HER2, triple negative), and the non-tumorigenic epithelial cell line MCF-10A.

[0078] The three CM tumor cell lines were cultured in Gibco Dulbecco's modified Eagle's medium (DMEM) high glucose (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 0.25 pg / ml amphotericin B, and 2 mM L-glutamine. MCF-10A cells were cultured in DMEM / F-12 medium (Nutrient Mixture) (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS, 1% penicillin-streptomycin, 0.25 pg / ml amphotericin B, 2 mM L-glutamine, 0.5 pg / ml hydrocortisone, 20 ng / ml epidermal growth factor (EGF), and 10 pg / ml insulin. All reagents used to supplement the culture medium were from Gibco, Carlsbad, CA, USA.All cell lines were cultured in a relative humidity atmosphere under standard temperature (37°C) and CO2 (5%) conditions and were grown in monolayers until they reached the optimal confluence and quantity for use in the different experiments. Cell lines were treated using concentrations of conglutins p 1, 3 and 6, diluted with culture medium, from 10 ng / pl to 2.5 ng / pl for 24 h.

[0079] Cellular irradiation

[0080] CM tumor cell lines were seeded in 6-well plates in the absence or presence of p-conglutin proteins. After 24 h, the culture medium was removed and replaced with fresh medium. Cells were then irradiated using X-ray equipment (Yxlon Smart Maxishoot 200-E, Copenhagen, Denmark) at room temperature (RT), under a constant current of 4.5 mA and 200 kV of power, achieving a radiation dose range of 0 Gy (control) to 8 Gy (0, 1, 2, 4, 6, and 8 Gy, respectively) using a field size of 15 cm x 8 cm with a focal length of 15 cm. Traceable dosimetry was performed following the calibration protocol, Technical Report Series (TRS) 398 of the International Atomic Energy Agency (IAEA).

[0081] Example 1: In vitro effect of conglutins P in combination with RL

[0082] To study the radiosensitizing effect of treatment with conglutin P proteins and their mechanisms of action, an in vitro CM model was used, in which treatments with individual conglutin P proteins were administered in combination with different radiation doses. A colony formation assay was performed, and dose-response curves were obtained for each cell line and experimental condition, allowing for estimation of cell survival in each case.

[0083] Colony formation assay

[0084] Irradiation was carried out following the previous cell irradiation protocol, with and without pretreatment with conglutin protein p for 24 hours. The concentrations of conglutin protein p used were 2.5 ng / ul for pi, 5 ng / ul for p3, and 10 ng / ul for p6. These doses were chosen to ensure that the viability of healthy MCF10-A epithelial cells was at least 85% after the treatments, and that the effect on the rest of the CM cells was significant enough

[0010] , The cells were seeded in 6-well plates with selected cell concentrations, from 500 to 4000 cells for MCF-7, from 1500 to 12000 cells for SK-BR-3 and from 1000 to 8000 cells for MDA-MB-231, for which a non-irradiated control and irradiation doses of 2, 4, 6 and 8 Gy were established. After the irradiation protocol, the cells were kept in the incubator for at least two weeks, depending on the growth for each cell line.

[0085] The culture medium was then removed and each well was stained with gentian violet dye (ICN Biomedicals, 101775) for 2–3 minutes. The wells were washed with water and the plates were left to dry overnight. The number of colonies was counted using an Olympus 1X51 optical microscope connected to an Olympus TH4-200 power microscope. A colony was defined as a cluster of cells with at least 50 cells and no signs of abortive colony (a non-homogeneous colony with holes in the center or any visual sign of cell death). All experiments were performed in triplicate for each condition. The cell survival fraction (SF) was calculated as follows:

[0086] FS = Number of colonies counted / (Number of cells seeded for this condition X cloning efficiency)

[0087] The obtained survival data were fitted to a linear-quadratic model, which provided the survival fraction and the cell survival curve parameters a and p as indicators of intrinsic radiosensitivity. The mathematical equation of the linear-quadratic (LQ) model is as follows:

[0088] FS = exp [ - (aD + D 2 )]

[0089] When assessing the results obtained in this test, it must be considered that simple treatment with conglutin p proteins, specifically with pi, p3 and p6, has a negative effect on the viability of CM cells

[0010] . This effect is not measured nor does it interfere in any way with the results of these tests, since the value measured at the end of the pretreatment with conglutin p proteins prior to starting radiation treatment is taken as the starting point to establish 100% survival. In this way, a control is taken as a starting point where the simple effect of the conglutin p proteins has already been subtracted. Therefore, the survival data of this test measure only the effect of radiation on CM cells, assessing in turn whether this effect is conditioned (enhanced or not) depending on the type of pretreatment with conglutin p proteins applied or its absence.For the following embodiments presented in this report, the design of the tests follows this same logic, so that the effect that the pre-treatment with conglutin proteins may have had on the CM cells is not taken into account, but rather whether this conditions to some extent the effectiveness of the subsequent treatment with RT.

[0090] The results of traceable dosimetry showed non-significant changes with respect to the resistance of conventional Rl treatment in healthy non-tumorigenic MCF-10A cells (Figure 3a), so we can conclude that pretreatment with conglutins does not negatively affect the survival of non-tumor cells under RT treatment, corroborating that conglutin p proteins are harmless to non-tumor cell lines at the concentrations studied

[0010] ,

[0091] The radiosensitizing effect of conglutin p proteins on tumor cell lines appears to differ depending on the conglutin p isoform and the tumor cell line type. In MCF-7 cells, pi significantly decreased the survival fraction for doses > 4 Gy (Figure 3b). In SK-BR-3 cells, pi significantly reduced (p<0.05) the survival fraction at 4 Gy, and pi, p3, and p6 reduced it at 6 and 8 Gy (Figure 3c). In the MDA-MB-231 cell line, the radiosensitizing effect was the greatest, with a significant reduction in the survival fraction starting at 2 Gy in experiments including conglutin pi, as well as a significant reduction in the survival fraction combining pi, p3, and p6 at higher radiation doses, from 4 to 8 Gy.In this case, for MDA-MB-231 cells, it is also important to mention that the same survival fraction is detected for the combined therapy (pi+2Gy) as that found for the single irradiation treatment at 4Gy (Figure 3d).

[0092] Based on these data, we can conclude that conglutins pi, p3 and p6 enhance the effect of Rl in vitro on BC tumor cell lines, especially pi on the tumor line corresponding to triple-negative BC, the most aggressive form of BC.

[0093] Example 2: In vitro effect of conglutins in combination with Rl on caspase-independent apoptosis.

[0094] The cytotoxic effect of the combined therapy of conglutins p and Rl was studied in the three tumor lines by evaluating the levels of apoptosis at 4 Gy and 6 Gy, doses at which the radiosensitizing effect was obtained in the three CM cell lines in Example 1, since at 2 Gy, only potential radiosensitization of MDA-MB-231 cells was found. Under these same experimental conditions (combined therapy of conglutins p and Rl in the three tumor lines at 4 Gy and 6 Gy, at the conglutin concentrations chosen in Example 1), the protein levels of total caspase 3, an executioner caspase, were studied by means of the Western Blot technique. Rl is known to activate the caspase cascade, which causes apoptosis of CM cells under normal conditions.This caspase activation has also been shown to stimulate tumor cell repopulation, which reduces the efficacy of long-term Rl treatments

[0012] . Thus, caspase-independent apoptosis induced by p-conglutins could reverse these unwanted effects.

[0095] Apoptosis assay

[0096] Apoptosis was analyzed using the IP-Annexin V kit (BD Biosciences, Franklin Lakes, NJ, USA). 2x10 5 cells / well in 6-well plates for each cell line and treated with p-conglutinins for 24 hours, in combination with the irradiation protocol, as previously described.

[0097] The samples were then trypsinized, washed, and incubated with AnnexinV-FITC and propidium iodide for 15 minutes in the dark. Samples were immediately analyzed using a BD FACS Aha lllu flow cytometer (Becton Dickinson, BD Bioscience). AnnexinV-FITC was detected using a blue laser (488 nm) FSC, with a 502 LP (Long Pass) filter and a 530 / 30 filter. Propidium iodide-PI was detected using a 561 YGL laser (561 nm), with a 600 LP and a 610 / 20 filter. Apoptosis was calculated by summing the apoptotic cells (AnnexinV-positive / Propidium iodide-negative population) and the late apoptotic cells (AnnexinV-positive / Propidium iodide-positive population).

[0098] Figure 4 shows how apoptosis increased significantly in all three cell lines, depending on the treatment with conglutin 3 and the dose of Rl. In MCF-7, conglutin 3 induced a higher apoptosis rate compared to Rl at 4 Gy (Figure 4a) and no significant differences were found (p<0.05) at 6 Gy, where all conditions showed extremely high apoptotic rates (>50%).

[0099] In SK-BR-3 cells, differences were found, although not statistically significant, for 3 to 4 Gy (Figure 4b), in agreement with what was shown in Figure 3, while i induced a significant peak of apoptosis compared to the control at 6 Gy.

[0100] Finally, in MDA-MB-231 cells (Figure 4c) pi and 3 induced 4 times more apoptosis in combination with a dose of 4 Gy compared to control cells, i showed a significant increase in the percentage of apoptosis at 6 Gy compared to the irradiated control.

[0101] To elucidate whether the increase in apoptosis is mediated by caspase activation, total caspase 3 protein levels were analyzed by immunoblotting at an Rl dose of 4 Gy (Figure 5) and 6 Gy (Figure 6) and using p-actin as a control.

[0102] The results showed that none of the three conglutin isoforms induced significant caspase 3 activation in any of the cell lines tested at 4 Gy (Figure 5). In fact, conglutin proteins p appear to reduce caspase 3 levels and apoptosis induction by p3 (MCF-7 cell line), and p1 and p3 (MDA-MB-231 cell line), meaning that both are caspase-independent. When the RI dose was increased to 6 Gy, p3 and p6 produced an increase in caspase 3 protein levels in all cell lines (Figure 6). This increase was not accompanied by an increase in the apoptosis rate (Figures 4a, 4b and 4c). The higher levels of apoptosis at 6 Gy induced by pi (SK-BR-3 and MDA-MB-231 cell lines) are also caspase-independent, since pi did not show any effect of increasing caspase 3 protein levels.

[0103] These higher apoptotic rates were not accompanied by caspase 3 activation, so that p-conglutins induce caspase-independent apoptosis not only in single treatment

[0010] , but also in combination with RT. Inhibition of caspase 3 does not compromise the therapeutic potential of RT and its cytotoxic effect

[0013] , It should be considered that defects in the signaling pathways leading to caspase activation are common in tumors, so that many tumor cells can unexpectedly survive caspase activation, so that caspase-independent cell death mechanisms are of great interest for cancer treatment

[0014] ,

[0104] Example 3: In vitro effect of conglutins in combination with Rl on CM cell lines.

[0105] To elucidate the effects on CM cell lines following combined treatment with conglutins p and Rl, intracellular ROS and DNA damage levels were measured following previously described methodologies.

[0106] Modulation of ROS in cancer cells may represent a potential strategy to overcome drug or radioresistance

[0015] , It is known that RI elevates intracellular levels of ROS, which have a dual role: they can trigger cell death in a p53-dependent manner and, in turn, increased ROS levels are also related to epithelial-mesenchymal transition (EMT) and carcinogenic transformation, being a potential initiating factor of malignant transformation

[0016] ,

[0107] Quantification of ROS

[0108] ROS levels were monitored by seeding 3 x 10 3cells / well in a 96-well plate and performing treatment with conglutin p proteins for 24 h in combination with the irradiation protocol described previously (CM cell lines with conglutins for 24 h followed by RI for 1 h using 4 and 6 Gy). The culture medium was then removed and the cells were washed twice with PBS and incubated with 100 µl of 10 pM 27'-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, St. Louis, MO, USA) in serum-free medium for 30 min in an incubator, in order to guarantee cell culture conditions. Finally, fluorescence was measured in a triad multimode reader using an excitation wavelength of 485 nm and an emission wavelength of 525 nm.Fluorescence results in relative fluorescence units (RFU) are understood to be comparable to intracellular ROS levels in cell lines, assuming higher levels for greater fluorescence [10, 17].

[0109] Combined treatment with conglutin py R1 induced lower overall ROS levels in CM cell lines compared to irradiated cells alone. A dysregulation of ROS levels was observed depending on the conglutin py isoform of the cell line type.

[0110] In the MCF-7 cell line, conglutin pi protein induced peak ROS levels at 4 Gy, whereas significant inhibition was observed at the same dose for p3 treatment. Combination therapy with p6 at 6 Gy of RI treatment decreased ROS levels. No significant effect was obtained when treatment was performed at 6 Gy with either conglutin pi or p3 isoforms (Figure 7a).

[0111] In SK-BR-3 cell lines, the combination of p3 and 4 Gy treatment exhibited an increase in ROS levels, while pi and p3 in combination with 6 Gy reduced ROS levels (Figure 7b).

[0112] In the MDAMB-231 cell line, no significant changes in ROS levels were found at 4 Gy, while a significant inhibition was found when cells were treated at the Rl dose of 6 Gy with all conglutins pi, and 3 and 6 (p < 0.05) (Figure 7c).

[0113] The results showed that ROS levels are treatment and dose dependent on Rl, presenting lower ROS levels under the same conditions where increased apoptosis was induced with the combined treatment. In some cases, conglutin p combined with Rl treatment induces peaks of ROS levels. Since the role of ROS is dual, those peaks could correspond to an accumulation of ROS leading to a switch from their carcinogenic functions to antitumor effects through the induction of cell death such as necrosis or ferroptosis

[0016] , The increased ROS levels in those specific cases showed a significant radiosensitizing effect in relation to the colony formation assay, so ROS could potentiate other caspase-independent cell deaths in those cases to obtain lower survival rates without an apoptotic increase rate.

[0114] The main mechanism of cell destruction induced by radiation is DNA damage, so DNA Damage Response (DDR) mechanisms are crucial for the radioresponse

[0018] , The sensitivity of cancer cells to radiation depends largely on their ability to recognize and respond to double-strand breaks (DSBs) produced by IRI. Some cancer cells, usually CSCs, promote radioresistance and survival by activating the DDR and DNA repair upon IRI exposure

[0019] ,

[0115] In this context, DNA damage was assessed after combined treatment of Rl with p-conglutinins by measuring phospho-yH2Ax by flow cytometry. The results of yH2Ax quantification can provide information about the DNA damage caused by Rl and the modification of the repair rate depending on the pretreatment. It has been shown that efficient DSB repair requires a coordinated DDR that includes phosphorylation of histone H2Ax, forming yH2Ax

[0020] , so its quantification not only provides information about the number of DSBs but also about the repair efficiency after treatment.

[0116] DNA damage

[0117] DNA damage in the different CM cell lines was quantified using the BD Cytofix / Cytoperm yH2Ax Detection Kit (BD Biosciences, Cat. 554714) and the PE-CF594 Mouse anti-H2Ax (pS139) antibody (BD Biosciences, Cat. 564719). In this case, 2 x 10 5cells / well in 6-well plates and then treated with the conglutin p proteins for 24 h in combination with the irradiation protocol as described for the rest of the experiments. After treatments, cells were trypsinized, washed twice, fixed and permeabilized using the Fixation and Permeabilization Buffer for 30 min at RT. Finally, cells were incubated with 100 pL of 1X BD Perm / Wash buffer for 15 min at RT and with the anti-H2Ax antibody for 30 min in the dark at RT. Samples were immediately analyzed by flow cytometry using the BD FACS Aha lllu flow cytometer (Becton Dickinson, BD Bioscience). For the detection of the yH2Ax population, the PETexas red fluorophore was used, a yellow-green laser 561 (561 nm), with a 600 LP and 610 / 20 filter.

[0118] Pretreatment with conglutins before Rl exposure dramatically reduced the phosphorylated yH2Ax population compared to the untreated Rl control, which promoted a significant reduction in the DNA repair rate after Rl. (Figure 8). Overall, the dysregulation of ROS levels was accompanied by a reduction in phosphorylated yH2Ax and an inhibition of the repair rate.

[0119] Treatment of the MCF-7 cell line with all three conglutin p isoforms induced a significant reducing effect on the levels of phosphorylated yH2Ax compared to control cells irradiated at 4 Gy, whereas p3 induced a significant reducing effect on yH2Ax levels when cells were treated with a higher R1 dose of 6 Gy (Figure 8a).

[0120] In the SKBR-3 cell line no significant changes were found for 4 Gy, but treatments with all isoforms of the conglutin p protein induced lower repair rates under 6 Gy (p < 0.05) in this cell line (Figure 8b).

[0121] Finally, in the MDA-MB-23 cell line, pi and p3 induced a significant decrease in yH2Ax at 4 Gy, while only pi combined with an Rl of 6 Gy induced decreased levels of yH2Ax (Figure 8c).

[0122] These results support a role for blue lupin seed conglutin-β proteins as inhibitors of natural repair rate, reducing the phosphorylated yH2Ax population upon exposure to RI and preventing the cell’s DNA repair mechanisms from overcoming the effect of RI, controlling tumor regrowth after treatment with conglutin-β. The combination of cytotoxic properties, apoptosis and caspase-independent killing, ROS modulation, and DNA repair inhibition confer to the antioxidant conglutin-β proteins their radiosensitizing role.

[0123] Example 4: Involvement of the SIRT1 / FoxO1 pathway and autophagy in the radiosensitization of CM cells under combined treatments of conglutin P and RL

[0124] In order to elucidate the possible mechanism of action of conglutins as a radiosensitizer in combination with RI, changes in the expression of SIRT 1 and FoxO1, as well as autophagy markers, were analyzed at both 4 Gy and 6 Gy.

[0125] The SIRT1 / FoxO1 regulatory axis is a ROS-sensitive pathway involved in the progression and aggression of BC [21-24]. Both its stimulation and inhibition are mechanisms of great interest in BC therapy, since this pathway shows a dual regulation depending on p53 and the phenotype of the tumor cell line.

[0126] Autophagy is one of the processes regulated by the SIRT1 / FoxO1 pathway, which is closely related to RT resistance. To evaluate autophagy, the proteins LC3B and p62, two autophagy markers, were studied after treatment. The role of autophagy with respect to RT could be dual, on the one hand, the inhibition of genes involved in autophagy results in tumor radiosensitization in vitro

[0025] ;but on the other hand, it induces radioresponse in vivo

[0026] since autophagy might provide an opportunity for cancer cells to survive in response to radiotherapy

[0027] , Recent evidence suggests that irradiation-induced cell death might be involved in autophagy [28, 29] and other studies suggest that autophagy might be an alternative of radiation-induced cell death in cancer, for those cells that present defects in the apoptosis pathway

[0030] , p53 has also been shown to be involved in radiation-induced autophagic cell death in BC

[0031] , Finally, a recent study showed that inhibition of autophagy sensitizes BC cells to radiation

[0032] , All these recent researches offer different possibilities for the role of autophagy in radioresponse in BC.;

[0127] Obtaining total proteins and Western Blot of cell cultures under different experimental conditions

[0128] Trypsinized cells were washed twice with ice-cold PBS (after treatments) and incubated with RIPA lysis buffer supplemented with protease inhibitors (Santa Cruz Biotechnology, Dallas, Texas, USA). Proteins were quantified by Bradford assay, denatured, and subsequently separated on SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to PVDF membranes using the Bio-Rad Trans Blot Turbo transfer system (Bio-Rad Laboratories, Inc., USA).

[0129] Membranes were then incubated overnight with appropriate primary antibodies against SIRT1, FoxO1, caspase 3, LC3B, p62, and p-actin (Abeam, Cambridge, UK) and subsequently with appropriate secondary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h. Finally, proteins were detected using Amersham ECL select Western Blot Detection Reagent (GE Healthcare, UK) on the membrane and images were acquired with the ChemiDoc MP Image Capture System (Bio-Rad Laboratories, Inc., USA). Western blot images were analyzed and quantified using Fiji software. Densitometry was performed for each membrane, and the area and mean intensity for each condition were calculated. Finally, the ratio of control actin to the protein of interest was calculated and compared with the ratio of the untreated control in each case [10, 17].

[0130] Following RI at 4 Gy, conglutin p protein isoforms induced a decrease in SIRT1 expression in all three cell lines studied (MCF-7, SK-BR-2, and MDA-MB-231). A parallel decrease in FoxO1 protein expression was also observed in MCF-7 and SK-BR-3 cell lines, whereas protein expression increased in MDA-MB-231 cells (Figure 9).

[0131] In contrast, at 6 Gy, conglutin p3 and p6 induced an increase in SIRT 1 expression in the MCF-7 cell line, and pi also increased SIRT 1 in MDA-MB-231 cells. The remaining treatments continued to induce lower levels of SIRT1 expression. These changes in SIRT1 expression at 6 Gy were accompanied by similar changes in FoxO1 expression (Figure 10).

[0132] The results suggest a dual effect of SIRT1 on radiosensitivity in CM and the important role of its activity through FoxO1.

[0133] Recent work has shown that targeting SIRT1 could be a promising therapy for radiosensitivity to RI, since its knockdown gene suppressed tumorigenesis and enhanced radiosensitivity in BC cell lines

[0033] , Controversially, several reports have shown that upregulation of SIRT1 could cause radiosensitivity in BC cells and reduce the CSC population, with an important role of IL-6, an interleukin that is regulated by β-conglutinins

[0034] , Moreover, its potential effect as a radiosensitizer has been demonstrated in BC cell lines despite its effect on SIRT 1 activation

[0035] ,

[0134] Thus, it is concluded that the final effect of treatment with conglutins as a radiosensitizer is strongly linked to a downregulation of the SIRT1 / FoxO1 pathway, to the status of p53, to the phenotypic characteristics of the cell line, to the radiation dose and to the radiosensitivity of each cell line used

[0036] ,

[0135] Regarding the role of autophagy, Figures 9 and 10 show the results at 4 Gy and 6 Gy respectively of the levels of LC3B and p62, autophagy markers, after treatment with conglutin proteins pi, p3 and p6.

[0136] In the MDA-MB-231 cell line at 4 Gy for the three conglutins p1, p3 and p6, an increase in LC3B levels and a decrease in p62 levels were observed. The same results were obtained for p6 at 6 Gy, suggesting that autophagy processes are activated under these conditions (conglutins p1 and R1 are dose-dependent).

[0137] In SKBR3 cells, decreased LC3B expression and increased p62 expression were observed at both doses of RI, demonstrating an inactivation of autophagy. No regulation of autophagy was observed in any case in MCF-7.

[0138] These results confirm the dual role of autophagy in cellular radiosensitization, depending mainly on the cell line, p53 status, regulation of the SIRT1 / FoxO1 pathway, and the cellular stress context (ROS and different doses of RI).

[0139] Example 5: In vitro effect of conglutins P in combination with Rl on CSCs.

[0140] Despite its therapeutic properties in BC, conventional RT has shown a potential increase in the CSC phenotype as an undesirable effect, which may be at the origin of tumor regrowth and metastasis as a late effect of RT [37, 38, 39]. Other studies have shown that RT failure could be attributed to the incomplete eradication of the CSC subpopulation [40, 41], so controlling the acquisition of the CSC phenotype and reducing this subpopulation would provide better clinical results and a reduction in side effects and relapse after exposure to RT.

[0141] Following treatment with conglutin p and Rl, breast CSCs were characterized using specific markers such as aldehyde dehydrogenase 1 (ALDH1) activity, which is directly related to CSC number, and the expression of surface markers CD44high / CD24low, and the results were compared with those of irradiated control cells without pretreatment with conglutin p proteins for each dose.

[0142] Quantification of ALDH1 positive enzymatic activity

[0143] The ALDEFLUOR™ kit (Stem Cell Technologies, Vancouver, Canada) was used to detect ALDH1 activity. After treatment, cells were trypsinized, washed, and incubated with BODIPY aminoacetaldehyde (BAAA), a non-toxic fluorescent substrate for ALDH, which was converted to BODIPY aminoacetaldehyde (BAA) and retained within the cells. Viable ALDH1+ cells were quantified by flow cytometry on a BD FACS Aha II lu flow cytometer (Becton Dickinson, BD Bioscience). The ALDH1-specific inhibitor, diethylaminobenzaldehyde (DEAB), was used to monitor background fluorescence. ALDH1-positive cells were quantified with a FITC fluorochrome that was detected using a blue laser (488 nm) FSC, with a 502 LP (Long Pass) filter and a 530 / 30 filter [10, 17],

[0144] ALDH1 activity was significantly decreased in MCF-7 at 4 Gy when cells were treated individually with conglutin isoforms pi, 3 and p6, respectively, whereas no significant differences were found at 6 Gy (Figure 11a).

[0145] Interestingly, when SK-BR-3 cells were treated with conglutin isoforms (Figure 11b), only 1pF at 4 Gy showed a significant increase in ALDH1 activity levels, which is consistent with the survival (Figure 3b) and apoptosis (Figure 4b) results at this dose of RI.

[0146] Finally, treatments in MDA-MB-231 with 3 and 6 reduce ALDH1 activity at 4 Gy, while only i induces those lower levels at 6 Gy (Figure 11c).

[0147] It is important to mention that ALDH1 activity was drastically reduced at 6 Gy compared to 4 Gy in control cells (not treated with conglutin P) for all three cell lines. Treatments with conglutin p3 and p6 in the MDA-MB-231 cell line at 4 Gy reduced ALDH1 levels as much as RI alone at 6 Gy, i.e., they showed similar ALDH1 percentages as the 6 Gy control (without treatment with conglutin P proteins); and treatment with p6 in MCF-7 at 4 Gy induced lower ALDH1 activity than in the 6 Gy control samples. Thus, depending on the cell line treated and the conglutin p protein isoform, combined treatment with RI may be more effective in decreasing the CSCs present in the tumor than increasing the RI dose alone.

[0148] Characterization of CSCs by flow cytometry

[0149] Phenotypic characterization of CSCs was performed using CD44-PE and CD24-FITC antibodies (Biolegend, San Diego, CA, USA). After treatments, cells were trypsinized, washed with cold PBS, and incubated for 30 minutes in the dark at 4°C with both antibodies. All samples were analyzed using a BD FACS Aria lllu flow cytometer (Becton Dickinson, BD Biosciences). The CD24-FITC antibody was detected using a blue laser (488 nm) FSC, with a 502 LP (Long Pass) filter and a 530 / 30 filter. CD44-PE was detected using a 561 YGL laser (561 nm), with a 582 / 15 filter [10, 17].

[0150] Similarly, conglutin-β treatments decreased the expression of the cell surface markers CD44high / CD24low in all three cell lines tested compared to control Rl cells. Specifically, all treatments induced a lower expression of the protein markers CD44high / CD24low in MCF-7 and SK-BR-3 cell lines at 4 Gy, whereas only conglutin 3 showed a significant decrease in these surface markers for MDA-MB-231 at 4 Gy.

[0151] In the MDA-MB-231 cell line, 6 Gy IR showed that all conglutin protein isoforms induce a decrease in the expression levels of CD44high / CD24low, but only conglutin p3 showed a significant decrease in these surface markers in the MCF-7 cell line, and the pi isoform in the SK-BR-3 cell line (Figure 12).

[0152] These results support that the combination of conglutins p and Rl regulates the potentiality phenotype in CM cells, inhibiting the ability of the CSC subpopulation to self-renew in vitro and preventing the acquisition of the potentiality phenotype that is uniquely associated with Rl therapy.

[0153] Example 6: In vitro effect of conglutins P in combination with Rl on self-renewal capacity.

[0154] The self-renewal capacity of CM cells was also analyzed after treatment with conglutin-p proteins followed by RI using the mammosphere assay. Mammosphere formation assay

[0155] 2x10 were planted 6cells / well in 6-well plates 2 days before starting the sphere formation assay and then treated for 24 hours with conglutins pi, p3 and p6, as previously described. The culture medium was then removed and the cells were irradiated following the cell irradiation protocol previously described and left for another 24 hours in the incubator. Once the desired treatment was carried out, the cells were trypsinized and seeded at a density of 1 x 10 2Cells / well were plated in triplicate in a 24-well ultra-low attachment plate (Corning, Massachusetts, USA) in DMEM / F12 supplemented with 1% P / S (penicillin / streptomycin), 10 pg / ml B27, 1 pg / ml hydrocortisone, 4 ng / ml heparin, 10 ng / ml EGF, and 20 ng / ml FGF. Spheres were then grown in the incubator for 5–8 days. Spheres larger than 50 pm in diameter were counted using a Leica DM500 binocular microscope [10, 17]. All reagents were purchased from Gibco (Carlsbad, CA, USA).

[0156] Conglutins pi, p3, and p6 significantly reduced the number of mammospheres in the MCF-7 cell line (Figure 13). pi and p3 reduced the number of mammospheres in the MDA-MB-231 cell line at both RI doses (4 and 6 Gy). Only conglutin pi in combination with 6 Gy was able to reduce the sphere-forming capacity of the SKBR-3 cell line. The characterization of these spheres by microscopy is shown in Figure 13d.

[0157] These results support the effect of conglutin p associated with Rl therapy in decreasing the self-renewal capacity of tumor cell lines and therefore support its role in preventing CM recurrence.

[0158] Statistical analysis

[0159] All experiments were performed at least in triplicate and results were expressed as mean ± standard deviation unless otherwise indicated. Statistical analyses were performed using the Shapiro–Wilk test to assess the normality of the data set and two-way ANOVA with Dunnett or Tukey correction using Graphad Prism 9 software (GraphPad software LLC, Boston, USA). Statistical differences between samples were considered significant when p values ​​were p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***).

[0160] Referencias 1. Sun, L. Radioresistance and Cancer Stem Cells: Survival of the Fittest. J Carcinog Mutagen s1 , (2011).

[0161] 2. Bhat, V. et al. Radiotherapy and radiosensitization in breast cancer: Molecular targets and clinical applications. Crit Rev Oncol Hematol 169, 103566 (2022).

[0162] 3. Jimenez-Lopez, J. C. Narrow-leafed lupin (Lupinus angustifolius L.) p-conglutin: A multifunctional family of proteins with roles in plant defence, human health benefits, and potential uses as functional food. Leg Sci 2, e33 (2020).

[0163] 4. Lima-Cabello, E. et al. Narrow-leafed lupin (Lupinus angustifolius L.) seed p-conglutins reverse the induced insulin resistance in pancreatic cells. Food Fund. 9, 5176-5188 (2018).

[0164] 5. Lima-Cabello, E. et al. Ex vivo and in vitro assessment of anti-inflammatory activity of seed p-conglutin proteins from Lupinus angustifolius. J Func Foods 40, 510-519 (2018).

[0165] 6. Lima-Cabello, E. et al. Narrow-leafed lupin (Lupinus angustifolius L.) p-conglutin proteins modulate the insulin signaling pathway as potential type 2 diabetes treatment and inflammatory-related disease amelioration. Mol Nutr Food Res 61 , (2017).

[0166] 7. Foley, R. C. et al. Analysis of conglutin seed storage proteins across lupin species using transcriptomic, protein and comparative genomic approaches. BMC Plant Biol 15, 106 (2015).

[0167] 8. Jimenez-Lopez, J.C, et al Lupin Allergy: Uncovering Structural Features and Epitopes of p-conglutin Proteins in Lupinus angustifolius L. with a Focus on Cross-allergenic Reactivity to Peanut and Other Legumes. In: Ortuño, F., Rojas, I. (eds) Bioinf and Biom Eng. vol 9043. Springer, Cham. (2015).

[0168] 9. Lima-Cabello E, Escudero-Feliu J, et a / .p-Conglutins' Unique Mobile Arm Is a Key Structural Domain Involved in Molecular Nutraceutical Properties of Narrow-Leafed Lupin (Lupinus angustifolius L). Int J Mol Sci. 2023 Apr 21 ;24(8):7676. (2023).

[0169] 10. Escudero-Feliu, J. et al. Narrow Leafed Lupin (Lupinus angustifolius L.) p-Conglutin Seed Proteins as a New Natural Cytotoxic Agents against Breast Cancer Cells. Nutrients 15, 523 (2023).

[0170] 9. Jimenez-Lopez, J. C. et al. Narrow-Leafed Lupin (Lupinus angustifolius L.) pi- and p6- Conglutin Proteins Exhibit Antifungal Activity, Protecting Plants against Necrotrophic Pathogen Induced Damage from Sclerotinia sclerotiorum and Phytophthora nicotianae. Front in Plant Sci 7, (2016).

[0171] 12. Rahmanian, N. et al. A. The paradox role of caspase cascade in ionizing radiation therapy. J Biomed Sci 23, 88 (2016).

[0172] 13. Rodriguez-Ruiz, M. E. et al. Apoptotic caspases inhibit abscopal responses to radiation and identify a new prognostic biomarker for breast cancer patients. Oncoimmunology 8, e1655964 (2019).

[0173] 14. Mathiasen, I. S. & Jááttelá, M. Triggering caspase-independent cell death to combat cancer. Trends in Mol Med 8, 212-220 (2002).

[0174] 15. Cui, Q. et al. Modulating ROS to overcome multidrug resistance in cancer. Drug Resist Updat 41 , 1-25 (2018).

[0175] 16. Wang, Y. et al. The double-edged roles of ROS in cancer prevention and therapy. Theranostics 11 , 4839-4857 (2021).

[0176] 17. Ríos-Arrabal, S. et al. Endothelin-1 as a Mediator of Heme Oxygenase-1-lnduced Sternness in Colorectal Cancer: Influence of p53. J Pers Med 11 , 509 (2021).

[0177] 18. Chen, Y. et al. LRRC31 inhibits DNA repair and sensitizes breast cancer brain metastasis to radiation therapy. Nat Cell Biol 22, 1276-1285 (2020).

[0178] 19. Deng, S. et al. Targeting the DNA Damage Response and DNA Repair Pathways to Enhance Radiosensitivity in Colorectal Cancer. Cancers (Basel) 14, 4874 (2022).

[0179] 20. Collins, P. L. et al. DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner. Nat Commun 11 , 3158 (2020).

[0180] 21. Hwang, J., et al. Redox regulation of SIRT1 in inflammation and cellular senescence. Free Radie Biol Med 61 , 95-110 (2013).

[0181] 22. Yao, H. & Rahman, I. Perspectives on translational and therapeutic aspects of SIRT1 in inflammaging and senescence. Biochem Pharmacol 84, 1332-1339 (2012).

[0182] 23. Shi, L. et al. A SI RT1 -centered circuitry regulates breast cancer sternness and metastasis. Oncogene 37, 6299-6315 (2018). 24. Fukui, M. et al. Mechanism underlying resveratrol’s attenuation of paclitaxel cytotoxicity in human breast cancer cells: Role of the SIRT1-FOXO1-HER3 signaling pathway. Cancer Treat Res Commun 28, 100386 (2021).

[0183] 25. Zhuang, W. et al. Induction of autophagy promotes differentiation of glioma-initiating cells and their radiosensitivity. Int J Cancer 129, 2720-2731 (2011).

[0184] 26. Ko, A. et al. Autophagy inhibition radiosensitizes in vitro, yet reduces radioresponses in vivo due to deficient immunogenic signalling. Cell Death Differ 21 , 92-99 (2014).

[0185] 27. Li H. et al. Autophagy-regulating microRNAs: potential targets for improving radiotherapy. J Cancer Res Clin Oncol. 144(9): 1623- 1634 (2018). 28. Kim, W. et al. Cellular Stress Responses in Radiotherapy. Cells 8, 1105 (2019).

[0186] 29. Levine, B. & Yuan, J. Autophagy in cell death: an innocent convict? J Clin Invest 115, 2679-2688 (2005).

[0187] 30. Kim, K. W. et al. Autophagy for cancer therapy through inhibition of pro-apoptotic proteins and mammalian target of rapamycin signaling. J Biol Chem 281, 36883-36890 (2006).

[0188] 31. Cui, L. et al. Radiation induces autophagic cell death via the p53 / DRAM signaling pathway in breast cancer cells. Oncol Rep 35, 3639-3647 (2016).

[0189] 32. Wang, B. et al. PLK1 Inhibition Sensitizes Breast Cancer Cells to Radiation via Suppressing Autophagy. Int J Rad One, Bio, Phy 110, 1234-1247 (2021).

[0190] 33. Zhang, X.,et al. . miR-22 suppresses tumorigenesis and improves radiosensitivity of breast cancer cells by targeting Sirtl . Biol Res 50, 27 (2017).

[0191] 34. Masoumi, H.,et al. The beneficial role of SIRT1 activator on chemo- and radiosensitization of breast cancer cells in response to IL-6. Mol Biol Rep 47, 129-139 (2020).

[0192] 35. Amini, P. et al. Resveratrol Induces Apoptosis and Attenuates Proliferation of MCF-7 Cells in Combination with Radiation and Hyperthermia. Current Mol Med 21 , 142-150 (2021).

[0193] 36. Mahmoud, A. S. et al. The Detection of DNA Damage Response in MCF7 and MDA- MB-231 Breast Cancer Cell Lines after X-ray Exposure. Genome Int (2023) doi:10.14293 / genint.14.1.001. 37. Kim, R.-K. et al. Radiation promotes malignant phenotypes through SRC in breast cancer cells. Cancer Sci 106, 78-85 (2015).

[0194] 38. Gomez-Casal, R. et al. Non-small cell lung cancer cells survived ionizing radiation treatment display cancer stem cell and epithelial-mesenchymal transition phenotypes. Mol Cancer 12, 94 (2013).

[0195] 39. Gao, X. et al. Radiation-Induced Reprogramming of Pre-Senescent Mammary Epithelial Cells Enriches Putative CD44+ / CD24- / low Stem Cell Phenotype. Front in One 6, (2016).

[0196] 40. Krause, M., et al. Cancer stem cells: targets and potential biomarkers for radiotherapy. Clin Cancer Res 17, 7224-7229 (2011).

[0197] 41. Pawlik, T. M. & Keyomarsi, K. Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol Biol Phys 59, 928-942 (2004).

Claims

CLAIMS 1. Conglutin p protein isolated from Lupinus angustifolius L. seeds for use as a radiosensitizer in radiotherapy.

2. The protein according to the preceding claim for use as a radiosensitizer in radiotherapy characterized in that it has an identity of at least 95% or at least 99% or 100% with SEQ ID NO 1 and has the activity and structural characteristics of the conglutin i protein, or an identity of at least 95% or at least 99% or 100% with SEQ ID NO 3 and has the activity and structural characteristics of the conglutin 3 protein, or an identity of at least 95% or at least 99% or 100% with SEQ ID NO 6 and has the activity and structural characteristics of the conglutin 6 protein.

3. The protein conglutin pi, 3 or 6 for use as a radiosensitizer in radiotherapy.

4. Composition comprising at least one protein according to any of claims 1 to 3 for use as a radiosensitizer in radiotherapy.

5. The composition according to the preceding claim which is a pharmaceutical composition.

6. The composition according to the preceding claim further comprising pharmaceutically acceptable excipients.

7. The protein according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 6 for use as a radiosensitizer in a method using radiotherapy for the treatment of cancer.

8. The protein according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 6 for use according to the preceding claim wherein the cancer is selected from the list comprising colon cancer, pancreatic cancer, hepatocarcinoma, head and neck cancer, bladder cancer, lung cancer and breast cancer.

9. The protein according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 6 for use according to the preceding claim wherein the cancer is breast cancer.

10. The protein according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 6 for use according to the preceding claim wherein the breast cancer is of the triple negative type.

Images