WO2014191608A1 - Peptides dérivés de la connexine 43 et composition pharmaceutique pour le traitement du cancer - Google Patents

Peptides dérivés de la connexine 43 et composition pharmaceutique pour le traitement du cancer Download PDF

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Publication number
WO2014191608A1
WO2014191608A1 PCT/ES2014/070444 ES2014070444W WO2014191608A1 WO 2014191608 A1 WO2014191608 A1 WO 2014191608A1 ES 2014070444 W ES2014070444 W ES 2014070444W WO 2014191608 A1 WO2014191608 A1 WO 2014191608A1
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Prior art keywords
peptide
cell
cells
stem cells
seq
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PCT/ES2014/070444
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English (en)
Spanish (es)
Inventor
Arantxa TABERNERO URBIETA
José María MEDINA JIMÉNEZ
Ester GANGOSO RODRÍGUEZ
Myriam JARAÍZ RODRÍGUEZ
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Universidad De Salamanca
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Publication of WO2014191608A1 publication Critical patent/WO2014191608A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a peptide comprising the amino acid sequence SEQ ID NO: 1, with the proviso that said peptide does not have the amino acid sequence SEQ ID NO: 2, and the use thereof for the treatment of diseases that They occur with cell proliferation, in particular, for the treatment of cancer and metastasis. Therefore, the present invention is included within the field of medicine, in particular, in the field of cancer treatment.
  • Gliomas are the most frequent brain tumors and generally have a very poor prognosis. In fact, patients diagnosed with a glioblastoma multiforme, the most frequent and severe type of glioma, have a half-life expectancy that does not exceed one year. Despite the advances in the study of this pathology and its treatment, this data has barely changed in recent years. The high degree of infiltration of these tumors makes their complete elimination in surgery very difficult and therefore recurrence is very frequent. Recently, a subpopulation has been identified in gliomas with characteristics similar to stem cells, called “glioma stem cells” (glioma stem cells, GSC) or "glioma initiating cells” (glioma initiating cells, GIC).
  • GSC glioma stem cells
  • GIC glioma initiating cells
  • glioma stem cells are responsible for the recurrence of gliomas. Due to resistance to conventional treatments, tumor stem cells persist after therapy and given their high oncogenic capacity cause tumor recurrence. For this reason, the cells Glioma mother have become a promising therapeutic target, since their removal or reversal of the stem cell phenotype would mean that all glioma cells would be susceptible to antitumor treatments.
  • the characteristics of the glioma stem cell phenotype is the expression of the transcriptional regulator Id1 (Soroceanu et al. Cancer Res.
  • Cx43 connexin43
  • Cx43 central nervous system
  • Gjs Gap junctions
  • Cx43 modifies the expression of several genes involved in cell cycle regulation, such as p21 and p27, two cyclin-dependent kinase inhibitors (CKI).
  • CKI cyclin-dependent kinase inhibitors
  • Patent application CN101966332 describes the use of "gap junction" proteins, in particular Cx43, to reverse the malignant phenotype of a carcinogenic stem cell.
  • Patent application CA2321976 describes the use of a retroviral vector to release a gap junction protein in cells, such as Cx43, restoring the expression of said gap junction proteins and treating So diseases like cancer.
  • Cx43 protein in clinics has the disadvantages that its solubilization in water at effective concentrations is low as well as its internalization and correct folding in the cell, which makes it necessary to use vectors and gene therapy for administration. To an individual.
  • patent application WO2007 / 084895 describes the use of the Cx43 terminal carboxyl end in the search for compounds or peptides capable of regulating Cx43 activity and its use in the treatment of arrhythmias and cancer.
  • the search for said compounds or peptides is a slow process, in which the peptides of interest are not always obtained, and if obtained, it is necessary to check their effect later, which can delay the effective treatment of tumors or cancer
  • the prior art there is a need to provide new alternative compounds to those described in the prior art, which have the same effects as the Cx43 protein in the reversal of the cell phenotype mother tumor and / or inhibition of cell proliferation, whose effectiveness in the treatment of tumors has already been proven, but that its administration does not present the drawbacks mentioned in previous paragraphs.
  • connexin-43 reverses the phenotype of the stem cells, in particular, human glioma stem cells, but until now it was unknown which part of the protein is responsible for that effect.
  • the inventors have discovered that said effect resides at the Cx43 terminal carboxyl terminus, namely, in the amino acid sequence SEQ ID NO: 1, and that peptides comprising said sequence of amino acids SEQ ID NO: 1 have the same effect as Cx43, being useful in the treatment of tumors or cancer.
  • the inventors electroplated human glioma stem cells with a vector containing Cx43, and observed that restoring Cx43 expression in said cells inhibited the activity of c-Src , the stem cell phenotype was reversed and their cell proliferation was inhibited. They also demonstrated that the effect of Cx43 on the glioma stem cell phenotype and on proliferation is triggered when Cx43 interacts with c-Src.
  • Another additional advantage is that using only the region responsible for reversing the phenotype of the tumor stem cell and / or inhibiting cell proliferation prevents the occurrence of unwanted effects by interaction of the rest of the Cx43 with other cellular effectors.
  • the use of peptides has the added advantage that it is possible to link cell internalization sequences to the peptides so that, when administered to an individual, they can be internalized in the cell to exert their effect without using therapy techniques. gene.
  • the present invention relates to a peptide comprising the amino acid sequence SEQ ID NO: 1 [AYFNGCSSPTAPLSPMSP] 1 or a functionally equivalent vain thereof with a sequence identity of at least 95% with the sequence SEQ ID NO: 1, hereinafter "peptide of the invention", with the proviso that said peptide does not have the amino acid sequence SEQ ID NO: 2.
  • Sequence SEQ ID NO: 2 shows the amino acid sequence SPSKDCGSPKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQA SEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNAKKVAAGHELQPL AIVDQRDA PRDASSED cd.
  • peptide is understood as that molecule formed by the union of between 15 and 150 amino acids by peptide bonds.
  • the peptide of the invention has a length of between 18 and 100 amino acids, preferably, between 18 and 50 amino acids, between 18 and 39 amino acids or between 18 and 20 amino acids.
  • the person skilled in the art will understand that, the smaller it is The peptide, the easier its administration will be and will have fewer side effects.
  • the peptide of the invention can be obtained by techniques widely known in the state of the art, such as chemical synthesis, genetic recombination, expression of the polynucleotide encoding the peptide of the invention, etc. All these techniques are routine practice for the person skilled in the art. Additionally, the carboxyl and amino terminal ends of the peptide of the invention may be protected against proteolysis. For example, the amino terminal end may be in the form of an acetyl group and / or the carboxyl end may be in the form of an amide group. It is also possible to carry out internal modifications of the peptides so that they are resistant to proteolysis.
  • the amino acids of the peptide of the invention may be in D-configuration, which may result in proteolysis resistant peptides.
  • the peptides can also be stabilized by intramolecular cross-linking, for example, by modifying at least two amino acid residues with oleophin side chains, preferably C3-C8 alkenyl chains, preferably, pentel-2-1 chains, followed by crosslinking. of chains as described in the technology called "staple" (Walensky et al., 2004, Science 205: 1466-1470). All of these chemically modified peptides to resist proteolysis are also contemplated within the present invention.
  • Additional modifications to the peptide of the invention comprise covalent binding to a polyethylene glycol (PEG) molecule by its terminal carboxyl end or to a lysine residue, in order to decrease its urinary elimination and therapeutic dose, and increase the half-life of the peptide in the blood plasma.
  • the half-life of the peptide can also be increased by including the peptide in a biodegradable and biocompatible polymeric material to form microspheres that are employed as a drug delivery system.
  • Polymers and copolymers include, but are not limited to, poly (D, L-lactide-co-glycolic) or PLGA.
  • the techniques and procedures of how to manufacture lipid microspheres or nanocapsules for use in the administration of drugs are widely known to those skilled in the art. Any method of administration of drugs selectively targeting a tumor population can be employed in the context of the present invention.
  • the peptide of the invention may comprise in its amino acid sequence conservative amino acid substitutions that do not vary the ability of the peptide to reverse the tumor stem cell phenotype and / or to inhibit cell proliferation, in particular, inhibit the proliferation of a tumor stem cell Therefore, in the context of the present invention, peptides derived from the peptide of the invention, referred to as "vanes", are also contemplated, although they do not have a sequence identity of 100% with the peptide of the invention, they retain their ability to inhibit cell proliferation, as amino acids have been replaced by biologically similar ones.
  • “functionally equivalent vain” is understood as that amino acid sequence with a sequence identity of at least 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 1 having the ability to reverse the tumor stem cell phenotype and / or inhibit cell proliferation, in particular, inhibit the proliferation of a tumor stem cell.
  • variants of the sequence SEQ ID NO: 1 are also included (such as the sequence SEQ ID NO: 23), as long as said fragments have the ability to reverse the tumor stem cell phenotype and / or inhibit cell proliferation , in particular, inhibit the proliferation of a tumor stem cell.
  • a fragment of the sequence SEQ ID NO: 1 is understood to be that amino acid sequence that has 100% identity with the sequence SEQ ID NO: 1 but lacks one or more amino acids of its amino- and / or carboxyl-terminus. terminal.
  • Example of an assay to find out if a peptide derived from the peptide of the invention has the ability to reverse the phenotype of a tumor cell and / or inhibit cell proliferation is described in Example 1.
  • identity or “sequence identity” is understood as the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity expressed as a percentage will be obtained.
  • the degree of identity between two amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as BLAST [Altschul S.F. et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215 (3): 403-10].
  • the BLAST programs for example, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are in the public domain on the website of The National Center for Biotechonology Information (NCBI).
  • any peptide that contains in its amino acid sequence SEQ ID NO: 1, or a functionally equivalent vain thereof with an identity of at least 95% with the sequence SEQ ID NO: 1, will have ability to reverse the phenotype of a tumor stem cell and / or to inhibit cell proliferation, in particular, to inhibit the cell proliferation of a tumor stem cell.
  • the peptide of the invention comprises the amino acid sequence SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 25: SEQ ID NO: 3 - DPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSP SEQ ID NO: 23 - FNGCSSPTAPLSPMSP
  • the peptide of the invention in order for the peptide of the invention to reverse the stem cell phenotype and / or inhibit cell proliferation, it is necessary for it to enter the cell and interact with the corresponding molecules, mainly c- Src
  • the introduction of the peptide into the cell can be done by any of the procedures known in the state of the art, such as direct injection, electroporation, transfection, etc. but these are relatively complex techniques with limitations when they have to be applied in vivo and access the entire population of tumor cells.
  • the peptide in case the peptide is to be administered to an individual, the peptide can be introduced into the cell by means of gene therapy techniques thanks to the use of viral vectors, or by means of cell internalization sequences that allow the peptide to cross the plasma membrane.
  • the peptide of the invention is covalently bound to an amino acid sequence of cellular internalization.
  • cell internalization amino acid sequence or “cell internalization sequence” or “cell penetration peptides” (CPPs) is understood as the amino acid sequences that possess the ability to transport molecules across the plasma membrane , without loss of integrity Among the most used sequences are TAT, Antennapedia (Antp) and oligo-arginines, whose characteristic in common is the presence of cationic amino acid groups. These internalization sequences allow internalization of the peptide directly to the cell. As the person skilled in the art understands, the cell internalization peptide can come from a natural source, or it can come from chemical synthesis and be an artificial sequence that does not exist in nature.
  • the cell internalization peptide may be linked to markers (for example, without limiting, fluorophores) that facilitate its localization.
  • markers for example, without limiting, fluorophores
  • cell internalization sequences include, but are not limited to, RQIKIWFQNRRMKWKK (SEQ ID NO: 7), VKKKKIKREIKI (SEQ ID NO: 8) [Guergnon J, et al. 2006. Mol Pharmacol. 69 (4): 1 1 15-24], FFLIPKG (SEQ ID NO: 9) [Ueda et al. 2012. Biomaterials, 35: 9061], SMoCs [Okuyama et al, 2007.
  • the cell internalization sequence comprises SEQ ID NO: 4 (YGRKKRRQRRR).
  • the cell internalization sequence may be linked to the peptide of the invention to both the amino terminal and the carboxyl terminal of the peptide.
  • the sequence of Cellular internalization is bound to the amino terminal end of the peptide.
  • peptides SEQ ID NO: 1 and SEQ ID NO: 3 can carry a cellular internalization sequence attached to their amino or carboxyl terminus. Therefore, in a particular embodiment, the peptide of the invention comprises the amino acid sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 24 or SEQ ID NO: 26.
  • the peptide of the invention can be obtained by techniques widely known in the state of the art, such as expression in a polynucleotide cell encoding the peptide of the invention, and its subsequent isolation. Therefore, in another aspect, the invention relates to a polynucleotide, hereinafter "polynucleotide of the invention", which encodes the peptide of the invention.
  • polynucleotide as used in the present invention, refers to a polymeric form of nucleotides of any length and formed by ribonucleotides and / or deoxyribonucleotides.
  • the term includes both single chain and double chain polynucleotides, as well as modified polynucleotides, that is, methylated, protected polynucleotides and the like.
  • the polynucleotide of the invention can be DNA, RNA or cDNA.
  • the invention relates to a gene construct, hereinafter "gene construct of the invention", which comprises the polynucleotide of the invention.
  • the gene construct of the invention comprises the polynucleotide of the invention operably linked to expression regulatory sequences of the polynucleotide of the invention.
  • any promoter can be used in the gene constructs of the present invention, provided that said promoter is compatible with the cells in which it is desired to express the polynucleotide.
  • a promoter, or promoter region is a nucleotide sequence that controls the transcription of a particular gene (nucleotide sequences). In the present invention it refers to a nucleotide sequence that controls the transcription of the polynucleotide of the invention.
  • Promoter sequences can be unidirectional or bidirectional.
  • a unidirectional promoter is one that controls the transcription of one gene or more genes that are placed in tandem with the first. "In tandem" means that the 3 'end of the first gene is followed, either consecutively or separated by a particular nucleotide sequence, by the 5' end of the second gene.
  • a bidirectional promoter refers to the promoter region that controls transcription in two opposite directions, for example the sequence that precedes the kivD gene of L.
  • lactis IFPL730 that acts as a bidirectional promoter. That is, a bidirectional promoter directs the transcription of two genes located divergently, that is in the opposite direction, the 5 'end of both nucleotide sequences being closer to each other than the 3' end.
  • promoter and “promoter region” are used interchangeably.
  • the promoters in the present invention can be constitutive or inducible.
  • inducible refers to the possibility that the promoter has a control element that allows activating or deactivating (repressing) the transcription of the gene it regulates, in the presence of a factor external to the promoter.
  • Promoters suitable for carrying out the present invention include, but are not limited to, constitutive promoters such as those derived from eukaryotic virus genomes such as polyoma virus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis virus B, the metallothionein gene promoter, the herpes simplex virus thymidine kinase gene promoter, retrovirus LTR regions, the immunoglobuin gene promoter, the actin gene promoter, the gene promoter EF-1 alpha as well as inducible promoters in which protein expression depends on the addition of an exogenous molecule or signal, such as the tetracycline system, the NFKB / light W system, the Cre / Lox system and the promoter of heat shock genes, the adjustable promoters of RNA polymerase II as well as tissue specific promoters. Promoters of glial cell specific genes include, but are not limited to, GFAP, nestin and s-100. Gl
  • the gene construct of the invention may contain markers or tags that allow isolation of the peptide of the invention once it is synthesized in the cell.
  • the polynucleotide or gene construct of the invention may be part of a vector.
  • the invention relates to a vector, hereinafter "vector of the invention", comprising the polynucleotide or gene construct of the invention.
  • vectors Suitable according to the present invention include, without limitation, (i) prokaryotic expression vectors such as pUC18, pUC19, Bluescript and its derivatives, mp18, mp19, pBR322, pMB9, ColEI, pCRI, RP4, phages and vectors " shuttle "such as pSA3 and pAT28, (i) yeast expression vectors such as 2 micron plasmid type vectors, integration plasmids, YEP vectors, centromeric plasmids and the like, (i ⁇ ) cell expression vectors of insects such as pAC series and pVL series vectors, (iv) plant expression vectors such as pIBI series, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB
  • the vector of the invention can be used to transform, transfect or infect cells capable of being transformed, transfected or infected by said vector.
  • Said cells can be prokaryotic or eukaryotic.
  • the vector where said DNA sequence is introduced can be a plasmid or a vector that, when introduced into a host cell, is integrated into the genome of said cell and replicated together with the chromosome / s in the / those that have been integrated.
  • the obtaining of said vector can be carried out by conventional methods known to those skilled in the art.
  • the invention relates to a cell, hereinafter "cell of the invention", which comprises a polynucleotide, a gene construct or a vector according to the present invention, for which said Cell has been able to be transformed, transfected or infected with the construct or vector provided by this invention.
  • Cell of the invention comprises a polynucleotide, a gene construct or a vector according to the present invention, for which said Cell has been able to be transformed, transfected or infected with the construct or vector provided by this invention.
  • Transformed, transfected or infected cells can be obtained by conventional methods known to those skilled in the art.
  • said host cell is an animal cell transfected or infected with an appropriate vector.
  • Suitable host cells for expression of the peptide of the invention include, without limitation, mammalian cells, plant cells, insect cells, fungal cells and bacterial cells.
  • Bacterial cells include, without limitation, Gram positive bacteria cells such as species of the genus Bacillus, Streptomyces and Staphylococcus and Gram negative bacteria cells such as cells of the genus Escherichia and Pseudomonas.
  • Fungal cells include, without limitation, yeast cells such as Saccharomyces, Pichia pastoris and Hansenula polymorpha, Aspergillus cells, and Trichoderma cells.
  • Insect cells include, without limitation, Drosophila cells and Sf9 cells.
  • Plant cells include, without limitation, crop plant cells such as cereals, medicinal, ornamental and bulb plants.
  • Mammalian cells suitable for the present invention include epithelial cell lines (pigs, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinomas (human, etc.).
  • glial cells (murine, etc.), hepatic cell lines (monkey, etc.), CHO cells (Ch ⁇ nese Hamster Ovary), COS cells, BHK cells, HeLa cells, 91 1, AT1080, A549, 293 or PER.C6 , human ECCs 5 NTERA-2 cells, D3 cells of the mESCs line, human embryonic stem cells such as HS293 and BGV01, SHEF1, SHEF2 and HS181, NIH3T3, 293T, REH and MCF-7 cells and hMSCs cells (human mesenchymal stem cells), and GNNS2 cells (glioma stem cells).
  • the inventors have discovered that the Cx43 terminal carboxyl terminus, namely, the amino acid sequence SEQ ID NO: 1, and peptides comprising said amino acid sequence SEQ ID NO: 1, are capable of reversing the phenotype of tumor stem cells and / or to inhibit cell proliferation, in particular, to inhibit the proliferation of tumor stem cells, such as glioma stem cells, which allows the use of the peptides of the invention in the treatment of tumors or cancer present in a subject.
  • tumor stem cells such as glioma stem cells
  • the invention relates to the use of a peptide, a polynucleotide, a gene construct, a vector or a cell of the invention in the preparation of a pharmaceutical composition.
  • a pharmaceutical composition The techniques and procedures for making pharmaceutical compositions are described below.
  • the invention relates to the use of a peptide, a polynucleotide, a gene construct, a vector or a cell of the invention in the preparation of a pharmaceutical composition for the treatment of benign or malignant tumors.
  • treatment is understood as the set of means used to treat, alleviate or cure a disease, in particular, tumors or cancer.
  • tumor means any growth of a tissue by uncontrolled cell proliferation.
  • the tumor can be benign or malignant.
  • a tumor is considered benign when the cells that form the tumor do not invade other tissues or cause metastases in other parts of the tumor. body.
  • the benign tumor is well encapsulated and the cells have no structure changes.
  • a tumor is considered to be malignant when the cells that form the tumor invade adjacent tissues, which is known as metastasis, and its cells have anaplasia.
  • malignant tumors are known as cancer.
  • the malignant tumor is cancer which, in another more particular embodiment, is a metastatic cancer.
  • the tumor or cancer (if it is malignant) can be located or originate in any tissue or organ. of the body.
  • any tumor or cancer is susceptible to being treated with the peptide of the invention, regardless of its state of development, its origin or its location.
  • cancer include, without limitation, lung cancer, colon cancer, skin cancer, pancreas cancer, stomach cancer, breast cancer, prostate cancer, liver cancer, cervical cancer, etc.
  • the tumor is a brain tumor (benign or malignant), that is, the brain is located.
  • the tumor is a glioma.
  • Glioma is a type of neoplasm that occurs in the brain or spinal cord. It is called glioma, since it arises from glial cells. Its most frequent location is the brain although it can also occur in the spinal cord. Any glioma can be treated with the pharmaceutical composition comprising the peptide, the polynucleotide, the gene construct, the vector or the cell of the invention.
  • Gliomas are named according to the specific type of cells that most closely resemble each other, classifying into ependymones (ependymal cells), astrocytomas (astrocytes) and oligodendrogliomas (oligodendrocytes), or according to the degree of tumor pathology, which may be low grade that is, gliomas they are well differentiated and are beningnos, or high grade, that is, gliomas are undifferentiated or anaplastic.
  • the glioma is a glioma selected from the group consisting of an astrocytoma, an oligodendroglioma and an ependymoma which, in an even more particular embodiment, the astrocytoma is a glioblastoma multiforme.
  • the tumor comprises stem cells, in particular tumor stem cells.
  • the peptide of the invention is capable of reversing the phenotype of tumor stem cells and / or inhibiting cell proliferation, in particular, inhibiting stem cell proliferation, more in in particular, tumor stem cells, even more particularly, tumor stem cells of glioma.
  • the invention relates to the use of a polynucleotide, a gene construct, a vector or a cell of the invention in the preparation of a pharmaceutical composition to reverse the phenotype of tumor stem cells and / or inhibit Cell proliferation, in particular, inhibit the proliferation of stem cells, more particularly, tumor stem cells, even more particularly, glioma tumor stem cells.
  • the invention relates to the use of a polynucleotide, a gene construct, a vector or a cell of the invention in the preparation of a pharmaceutical composition to prevent cell proliferation and / or prevent cancer metastasis.
  • the prevention of cell proliferation comprises the prevention of cell proliferation of stem cells, more particularly, tumor stem cells, even in particular, glioma stem cells.
  • the invention relates to the use of a peptide, a polynucleotide, a gene construct, a vector or a cell of the invention as a reagent to reverse in vitro the phenotype of tumor stem cells and / or inhibit in vitro cell proliferation.
  • the prevention of cell proliferation comprises the prevention of cell proliferation of stem cells, more particularly, tumor stem cells, even in particular, glioma tumor stem cells.
  • a tumor stem cell whose phenotype has been reversed is characterized by the high expression of Id1, Sox2 and N-caderina, the low expression of Cx43 and E-caderina, and the high activity of c-Src.
  • a peptide has the ability to reverse the tumor stem cell phenotype" when said peptide when coming into contact with tumor stem cells causes said cells to lose the mentioned phenotypic characteristics.
  • Example of an essay for checking whether the phenotype of a tumor stem cell has been reversed is, for example, an immunocytochemical study, by PCR or by Western blot of the expression of Id1, Sox2, N-caderin, E-caderin or any other stem cell marker .
  • inhibiting cell proliferation is understood as the reduction, decrease, attenuation or blockage of cell division or cycle.
  • An assay to verify that cell proliferation of a cell has been inhibited is, for example, the MTT colorimetric assay as described in Example 1 herein.
  • subject is understood as any animal, preferably a mammal, more preferably a primate, in particular, a human being, of any race, sex or age.
  • the invention relates to the use of a peptide, a polynucleotide, a gene construct, a vector or a cell of the invention as a reagent to inhibit in vitro the activity of the protein kinase c-Scr or the activity of the transcriptional regulator Id1.
  • the invention relates to the use of a peptide, a polynucleotide, a gene construct, a vector or a cell of the invention for the in vitro identification of tumorigenicity regulating or modulating compounds.
  • Pharmaceutical Composition of the Invention As explained in the previous inventive aspect, the peptide, the polynucleotide, the gene construct, the vector or the cell of the invention can be used in the preparation of a pharmaceutical composition.
  • the invention relates to a pharmaceutical composition, hereinafter, pharmaceutical composition of the invention, comprising a peptide, a polynucleotide, a gene construct, a vector or a cell of the invention in a therapeutically effective amount, and a pharmaceutically acceptable carrier.
  • pharmaceutical composition or “medicament” means any preparation or pharmaceutical form, whose formula of composition expressed in units of the international system, is constituted by a substance or mixture of substances, with constant weight, volume and percentages , prepared in legally established pharmaceutical laboratories, packaged or labeled to be distributed and marketed as effective for diagnosis, treatment, mitigation and prophylaxis of a disease, physical anomaly or symptom, or the restoration, correction or modification of the balance of the organic functions of the human beings and animals.
  • the preparation of the pharmaceutical composition can be carried out by any of the methods described in the state of the art.
  • the dosage to obtain a therapeutically effective amount depends on a variety of factors, such as age, weight, sex or tolerance, of the mammal.
  • the term “therapeutically effective amount” refers to the minimum amount of the compound or pharmaceutical composition of the invention necessary to produce the desired effect and, in general, will be determined, among other factors, by the characteristics of said compound or said pharmaceutical composition and the therapeutic effect to be achieved.
  • adjuvants and “pharmaceutically acceptable carriers” that can be used in the compositions of the invention are widely known in the state of the art.
  • the term "vehicle” refers to a diluent or excipient with which the active ingredient is administered.
  • Such pharmaceutical vehicles may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, or may come from a natural source or be a diluent or excipient that does not exist naturally.
  • water or aqueous solutions of saline solution and aqueous solutions of dextrose and glycerol are used as vehicles, particularly for injectable solutions.
  • the vehicles of the invention are approved by a state or federal government regulatory agency or are listed in the United States Pharmacopoeia or other pharmacopoeia generally recognized for use in animals, and more particularly in humans.
  • the vehicles and auxiliary substances necessary to manufacture the desired pharmaceutical form of administration of the pharmaceutical composition of the invention will depend, among other factors, on the pharmaceutical form of administration chosen.
  • Said pharmaceutical forms of administration of the pharmaceutical composition will be manufactured according to conventional methods known to those skilled in the art.
  • compositions of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways known in the state of the art.
  • they can be, without being limited to, in aqueous or non-aqueous solutions, in emulsions or in suspensions.
  • non-aqueous solutions include, not limited to, propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • aqueous solutions include, but are not limited to, water, alcoholic solutions in water and saline media.
  • Aqueous solutions may be buffered or not, and may have additional active or inactive components.
  • Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, or the like, or nutrients, including glucose, dextrose, vitamins and minerals.
  • preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, or the like, or nutrients, including glucose, dextrose, vitamins and minerals.
  • nutrients including glucose, dextrose, vitamins and minerals.
  • the compositions can be prepared for administration in solid form.
  • compositions may be combined with various inert carriers or excipients, including but not limited to: binders, such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients, such as starch or lactose; dispersing agents, such as alginic acid or corn starch; lubricants, such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or flavoring agents, such as peppermint or methyl salicylate.
  • binders such as microcrystalline cellulose, gum tragacanth, or gelatin
  • excipients such as starch or lactose
  • dispersing agents such as alginic acid or corn starch
  • lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide
  • sweetening agents such as sucrose or saccharin
  • flavoring agents such as peppermint or methyl salicylate.
  • composition of the invention may comprise an adjuvant.
  • adjuvant is meant any substance that enhances the effectiveness of the pharmaceutical composition of the invention.
  • adjuvants include, but are not limited to, adjuvants formed by aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc., oil-in-water or water-in-oil emulsion formulations such as the adjuvant Complete Freunds (CFA) as well as the incomplete Freunds Adjuvant (IFA); mineral gels; block copolymers, Avridine TM, SEAM62, adjuvants formed by components of the cell wall of bacteria as adjuvants that include liposacahydes (eg lipid A or Lipido A monophosphohl (MLA), trehalose dimicolate (TDM) and skeletal components of the cell wall (CWS), heat shock proteins or their derivatives, adjuvants derived from bacterial toxins ADPribosilatinadas, including diph
  • sphaerieus toxin C. botulinum C2 and C3 toxins, C. limosum exoenzyme as well as C. perfringens, C. spiriforma and C. diffieile, S. aureus, EDIM toxins and mutant toxin mutants such as CRM-197, a non-toxic mutant toxin from diphtheria; saponins such as ISCOMs (immunostimulatory complexes), chemokine kemokines and cytokines such as interleukins (for example, IL-I IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12 , etc.), interferons (e.g., interferon gamma) macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), defensins 102, RANTES, MIPI-alpha.
  • ISCOMs immunonostimulatory complexes
  • muramyl peptides such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L- alanyl- Disoglutaminyl- L- alanine-2- (1 '-2'-dipalmitoyl-s- n-glycero-3 huydroxyphosphoryloxy) ethylamine (MTP-PE), etc; adjuvants derived from the family of CpG molecules, CpG dinucleotides and synthetic oligonucleotides comprising CpG motifs, lisosum exoenzyma of C.
  • thr-MDP N-acetyl normuramyl-L-alanyl-D-isoglutamine
  • nor-MDP N-
  • Limosum and synthetic adjuvants such as PCPP, cholera toxin, Salmonella toxin, alum and the like, aluminum hydroxide, N- acetyl-muramyl-Ltreonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, dimicolate of trehalose and cell wall skeleton (MPL + TDM + CWS) in a 12% squalene emulsion / Tween 80.
  • Other examples of adjuvants include, but are not limited to, DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants and QuilA.
  • the pharmaceutical composition of the invention further comprises a chemotherapeutic agent.
  • chemotherapeutic agent any substance that is capable of inhibiting cell proliferation without necessarily killing the cell, or that is capable of inducing cell death.
  • cytostatic agents agents capable of inhibiting cell proliferation without causing cell death
  • cytotoxic agents those that are capable of inducing cell death normally by activating apoptosis
  • Non-limiting examples of chemotherapeutic agents suitable for use in the compositions of the invention include, but are not limited to, (i) microtubule stabilizing agents such as taxanes, paclitaxel, docetaxel, epothilones and laulimalides, (i) kinase inhibitors such as Iressa (R), Gleevec, Tarceva TM, (Erlotinib HCI), BAY-43- 9006, (iii) specific antibodies to receptors with kinase activity including, but not limited to, Trastuzumab (Herceptin (R)), Cetuximab (Erbitux (R) )), Bevacizumab (Avastin TM), Rituximab (ritusan (R)), Pertuzumab (Omnitarg TM); (iv) mTOR pathway inhibitors, such as rapamycin and CCI-778; (v) Apo2L1 Trail, (vi) anti-angi
  • compositions and / or their formulations may be administered to an animal, including a mammal and, therefore, to man, in a variety of ways, including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intrathecal, intraventricular, intraarticular. , intratumoral, oral, enteral, parenteral, intranasal, ocular or topical.
  • a preferred route of administration of the compositions and / or formulations of the compound of the invention for the prevention or treatment of a cancer is the intratumoral route.
  • the pharmaceutical composition of the invention is formulated for oral, parenteral, nasal or sublingual administration.
  • Kit of the invention Administration of the peptide of the invention requires a series of components that can be arranged together in the form of a pack or kit.
  • kit of the invention comprising a peptide, a polynucleotide, a gene construct, a vector or a cell of the invention described in the above inventive aspects together to their corresponding particular embodiments.
  • Components useful for administration of the peptide of the invention and which may be comprised within the kit include, but are not limited to, buffer solution, lysis solution, sterile material (syringes, swabs, swabs, tweezers, etc.), distilled water, alcohols (ethanol), etc. Additionally, the kit may contain instructions or indications that guide the person skilled in the art in administering the peptide of the invention.
  • the invention relates to the use of a kit for the determination of the effect of said peptide of the invention on the tumorigenicity of a cell line, to inhibit cell proliferation in vitro and / or reverse the cell phenotype in vitro. mother tumor, or to inhibit in vitro the activity of protein kinase c-Scr or the activity of the transcriptional regulator Id1.
  • the prevention of cell proliferation comprises the prevention of cell proliferation of stem cells, more particularly, tumor stem cells, even in particular, glioma stem cells.
  • the invention relates to a method for the treatment and / or prevention of tumors in a subject, both benign and malignant (cancer or metastasis), which comprises administering to said subject a peptide, a polynucleotide, a gene construct, a vector or a cell according to the invention.
  • the invention also relates to a method of inhibiting (in vitro / in vivo) cell proliferation and / or reversing the tumor stem cell phenotype, as well. how to inhibit (in vitro / in vivo) the activity of the c-Scr protein kinase or the activity of the transcriptional regulator Id1.
  • the invention is also directed to a method for the in vitro identification of tumorigenic regulatory or modulatory compounds.
  • FIGURES Figure 1 Dose response of Cx43 on the activity of c-Src in GMNS2 glioma stem cells.
  • GNNS2 glioma stem cells were transfected with different concentrations (0.5, 1, 1, 5 and 2 pg / pL) of Ires or Ires-Cx43 plasmid and the proteins were collected at 48h after transfection.
  • A-actinine was used as a load control.
  • FIG. 1 Effect of Cx43 restoration on activity and c-Src on GMNS2 glioma stem cells.
  • GIINS2 glioma stem cells were transfected with 1 g / L of Ires or Ires-Cx43 plasmid and proteins were collected 2 and 5 days after transfection.
  • the values of c-Src Y416 (b) and total c-Src (c) were quantified and normalized with ⁇ -actinin.
  • the significance of the differences with respect to the control situation are expressed as ** p ⁇ 0.01 (Student's t test).
  • FIG. 3 Effect of the restoration of Cx43 on the growth of GMNS2 glioma stem cells.
  • GNNS2 glioma stem cells were transfected with I pg / pL of plasmid Ires or Ires-Cx43.
  • b) From 3 h after transfection and for 7 days, the number of viable cells per plate was measured, using the MTT method, as described in Material and Methods. The results are expressed as a percentage of the maximum absorbance value at day 7 and are mean + SEM (n 4). The significance of the differences with respect to the control situation is expressed as ** p ⁇ 0.01 (Student's t test).
  • FIG. 4 Effect of the restoration of Cx43 on the expression of Ki-67 protein in GMNS2 glioma stem cells.
  • FIG. 5 Effect of Cx43 on the expression of Sox2, E-cadherin and N-cadherin in the GMNS2 glioma stem cell line.
  • GNNS2 glioma stem cells were transfected with I pg / pL of plasmid Ires or Ires-Cx43. They were grown in conditions and stem cell. Cells were processed for Western blot analysis or fixed for immunocytochemistry, 5 days after transfection. a) Phase contrast photomicrographs and immunocytochemistry of Sox2 transfection b) Double immunocytochemistry of Sox2 and Cx43 in cells transfected with the plasmid Ires-Cx43.
  • GNNS2 glioma stem cells were transfected with I pg / pL of plasmid Ires or Ires-Cx43. They were grown in stem cell medium. Cells were processed for Western blot analysis or fixed for immunocytochemistry, 5 days after transfection.
  • FIG. 7 Effect of c-Src inhibition on the expression of Id1 in GMNS2 glioma stem cells.
  • GNNS2 glioma stem cells were reseeded at a density of 50,000 cells / cm 2 and cultured in the presence of the indicated concentrations of c-Src, Dasatinib, Saracatinib and PP2 inhibitors. Always using the same concentration of DMSO as a control, used as a vehicle. After 24 or 48 hours, proteins were collected and analyzed by Western blot analysis, a) Western blot Id1, c-Src Y416, total c-Src and ⁇ -actinine. b) Western blot of Id1 and a-actinin.
  • Figure 8 Effect of c-Src inhibition on the expression of Sox2, E-cadherin and N-cadherin in GMNS2 glioma stem cells.
  • GNNS2 glioma stem cells were reseeded at a density of 50,000 cells / cm 2 and cultured in the presence of the indicated concentrations of c-Src, Dasatinib, Saracatinib and PP2 inhibitors. Always using the same concentration of DMSO as a control, used as a vehicle. After 24 or 48 hours, proteins were collected and analyzed by Western blot analysis.
  • FIG. 9 Effect of the TAT-Cx43Src peptide on the expression of Sox2, Id1, N-cadherin, E-cadherin, c-Src Y416 and total c-Src in GMNS2 glioma stem cells.
  • GNNS2 stem cells were reseeded at a density of 50,000 cells / cm 2 and incubated in the presence of TAT or TAT-Cx43Src peptides at the concentration of 50 ⁇ . Samples were collected at 24 and 48 hours and analyzed by Western blot, a) Sequence of the penetrating peptide containing the region of Cx43 that interacts with c-Src.
  • the TAT sequence is YGRKKRRQRRD (SEQ ID NO: 4), the tyrosines phosphorylated by c-Src appear in bold and larger, the SH3 binding region of c-Src appears underlined, b) Phase contrast photomicrographs and immunocytochemistry of Cx43 c) Western blot of Sox2, Id1, N-cadherin, E-cadherin, c-Src Y416, total c-Src and a-actinine.
  • FIG. 10 Effect of the TAT-Cx43S peptide on the expression of Sox2, Id1, N-cadherin, E-cadherin and c-Src Y416 in GMNS2 glioma stem cells.
  • GNNS2 stem cells were reseeded at a density of 50,000 cells / cm 2 and incubated in the presence of the TAT or TAT-Cx43S peptides at the concentration of 50 or 100 ⁇ . Samples were collected at 24 hours and analyzed by Western blot, a) Sequence of the penetrating peptide containing the region of Cx43 that interacts with c-Src.
  • the TAT sequence is YGRKKRRQRRD (SEQ ID NO: 4), the SH3 binding region of c-Src is underlined, b) Western blot of N-cadherin, E-cadherin, c-Src Y416 and ⁇ -actinine. c) Western blot of Sox2, Id1, c-Src Y416, and a-actinine
  • FIG. 13 Effect of TAT-Cx43Src, TAT-Cx43S and TAT-SH3 (SEQ ID NO: 22) on the expression of Sox2, Id1 and c-Src Y416 in GMNS2 glioma stem cells.
  • Figure 14 Effect of the different penetrating peptides based on the Cx43 region that interacts with c-Src on the proliferation of G166 glioma stem cells.
  • FIG. 15 Effect of TAT-Cx43Src, TAT-Cx43S and TAT-SH3 (SEQ ID NO: 22) on the expression of Sox2, Id1 and c-Src Y416 in GMNS2 glioma stem cells.
  • GNNS2 glioma stem cells were seeded at a density of 1 or 2 cells per well in 96-well plates and incubated in the presence of TAT, TAT-Cx43Src, TAT-Cx43S and TAT-SH3 (SEQ ID NO: 22 ) at a concentration of 50 ⁇ . After 4 weeks the number of neurospheres was determined. ** p ⁇ 0.01 shows the significant differences compared to the control.
  • B) GNNS2 glioma stem cells were incubated in the presence of the indicated peptides at a concentration of 25 ⁇ for 96 hours. O4 immunocytochemistry and DAPI nuclear staining of the same field.
  • Neurons were cultured as described in Material and Methods. The indicated peptides were added to the culture medium at a concentration of 50 ⁇ . Phase contrast photomicrographs were taken 24 and 48 hours after peptide addition. No differences are observed either in the number of cells or in cell morphology.
  • G166 cells were added per well of 1.9 cm 2 .
  • the corresponding peptides were added and at 80 hours the cells were fixed, immunocytochemicals were carried out against human Nestine to identify G166 cells and the G166 nuclei and astrocytes were stained with DAPI.
  • the decrease of G166 cells is observed after treatment with TAT-Cx43S.
  • GSC Human glioma stem cell
  • glioma stem cell lines characterized and recently deposited in Biorep have been used (Steve Pollard, Austin Smith, Peter Dirks and lan Clarke).
  • the human glioma stem cells G144, G166, GNNS2 and G179 have been purified from glioblastomas after surgery in four different patients. These cells have the characteristics of human glioma stem cells, including the capacity for self-renewal, differentiation in different neural lineages, resistance to conventional therapies and most importantly, they have a high capacity to generate very aggressive tumors, similar to the original ones in the patients, when they are xenotransplanted in immunosuppressed mice.
  • the cells have been cultured in neurobasal culture medium supplemented with 2% B27, 1% N2, 20 ng / ml EGF and 20 ng / ml bFGF, in laminin-coated culture plates, to generate adherent cell cultures. Thanks to their capacity for self-renewal, when these cells expand in adherent cultures under these culture conditions, they maintain the aforementioned properties, that is, the GSC cararteristic phenotype and genotype, including its tumorigenicity, for more than 20 passes.
  • the method commonly used in the laboratory has been followed (Herrero-González et al., 2010, cited ad supra).
  • the connexin cDNA43 was obtained from the human glioma line GI ⁇ Ns2 and was cloned into the bicistronic plasmid plRESpuro2 (Clontech) that provides puromycin resistance.
  • the Sequence obtained was confirmed in the automatic sequencing service of the University of Salamanca.
  • Transfection of the empty vector (Ires) or of the vector containing the Cx43 (Ires-Cx43) in the human glioma stem cells GI ⁇ Ns2 was performed by electroporation. Transfected cells were selected with 0.1 mg / ml of puromycin.
  • Transfection of the empty vector (Ires) or of the vector containing the Cx43 (Ires-Cx43) in the human glioma stem cells GI ⁇ Ns2 was performed by electroporation. Transfected cells were selected with 0.1 mg / ml of puromycin.
  • Two peptides were designed based on the region of Cx43 that interacts with c-Src.
  • the first one comprises the sequence of union of Cx43 with the SH3 domain of c-Src [SEQ ID NO: 1 (AYFNGCSSPTAPLSPMSP)] and the second, also comprises the region of Cx43 that includes the two tyrosines phosphorylated c-Src [SEQ ID NO: 3 (DPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSP)] (tyrosines are marked in bold).
  • TAT sequence of cellular internalization SEQ ID NO: 4 [YGRKKRRQRRR].
  • the final result was two internalizing sequences based on the junction region of Cx43 with c-Src (SEQ ID NO: 5) and on the junction region of Cx43 with c-Src together with the region of Cx43 phosphorylated by c -Src (SEQ ID NO: 6).
  • the internalization of the peptides was achieved by solubilizing them in water and adding them to the culture medium at 37 ° C for 24 or 48 hours. These peptides were used at concentrations of 50-100 ⁇
  • c-Src The activity of c-Src depends on its autophosphorylation in Tyr416 which increases its catalytic activity. On the other hand, phosphorylation in Tyr527 inhibits its activity. Therefore, to determine the activity of c-Src, phosphorylated c-Src levels were analyzed in Tyr416 and Tyr527. Western type transfer analysis.
  • the "Western" type transfer analysis was performed following the method commonly used in the laboratory. Briefly, it is about homogenizing the cells in culture, in Tris buffer containing SDS, EDTA, EGTA and a mixture of protease and phosphatase inhibitors. Electrophoresis was performed in SDS-polyacrylamide gels, the proteins were transferred to nitrocellulose membranes where immunodetection was performed. The membranes were incubated with the desired primary antibody followed by a secondary antibody labeled with peroxidase. The detection was carried out by chemiluminescence following the manufacturer's instructions (Luminol; Santacruz).
  • the antibodies used and the dilutions were the following: Cx43 (Transduction Laboratories 610062, 1: 250), Y416-Src (Cell Signaling Technology 2101, 1: 250), total-Src (Cell Signaling Technology 2108 and 21 10, 1: 500 ), Id1 (Santa Cruz Biotechnolgy Inc. sc-488, 1: 500), Sox2 (Abcam ab97959, 1: 1000), E-cadherin (S Santa Cruz Biotechnolgy Inc sc-7870, 1: 100), N-cadherin ( Santa Cruz Biotechnolgy Inc sc-7939, 1: 500). Antibodies against GAPDH (Applied Biosystems AM4300, 1: 5000) or ⁇ -actinin (Merk Millipore MAB1682, 1: 1000) were used as load control.
  • Cx43 Transduction Laboratories 610062, 1: 250
  • Y416-Src Cell Signaling Technology 2101
  • the cells were fixed with 4% paraformaldehyde, for 20 min. After successive washing with PBS, the cells were incubated with the primary antibody.
  • the dilutions used and the characteristics of the antibodies were the following: Cx43 (Transduction Laboratories 610062, 1: 200), Id1 (Santa Cruz Biotechnology Inc. sc-488, 1: 500), Sox2 (Abcam ab97959 1: 500), Ki -67 (Sigma P6834, 1: 200), N-cadherin (Santa Cruz Biotechnology Inc sc- 7939, 1: 200).
  • the cells were washed with PBS and incubated with the fluorescent-conjugated secondary antibody, diluted in PBS containing 10% FCS, 0.1 M glycine and 0.02% sodium azide. After 1 hour the cells were washed with PBS and mounting medium was added for observation in an inverted fluorescence microscope or in a confocal microscope, with the appropriate filters. In all cases, controls were carried out using exclusively the secondary antibody.
  • the cells were seeded at low density and the number of viable cells was determined throughout the different days in culture.
  • the culture medium was removed and the cells were incubated with 300 ⁇ of PBS in the presence of MTT (0.5 mg / ml), for 75 minutes, in the dark, at 37 ° C, in a CO2 incubator.
  • MTT 0.5 mg / ml
  • the medium was subsequently removed, and 500 ⁇ of DMSO was added.
  • the cells were shaken in darkness for 10 minutes. Finally, the absorbance at 570 nm was determined.
  • gliomas are composed of a heterogeneous population of cells, some of them with stem cell characteristics, these are the so-called glioma stem cells. These cells are characterized by their capacity for self-renewal, the generation of multiple cell types, their high oncogenic potential, and resistance to conventional treatments.
  • Cx43 reduces the oncogenic activity of cSrc in C6 rat glioma cells, therefore, in this work we wanted to investigate, first of all if Cx43 also affects c-Src activity in glioma stem cells .
  • GNNS2 glioma stem cells were transfected by electroporation with different concentrations of the Ires-Cx43 construct or with the empty Ires vector. Subsequently, the samples were analyzed by the Western blot technique.
  • Figure 1 shows an increase in Cx43 expression, as the concentration of plasmid lresCx43 increases.
  • Figure 3B shows that cells transfected with Cx43 (Ires-Cx43) have a growth curve analyzed by MTT, smaller than cells transfected with the empty plasmid (Ires), this difference being highly significant after 5 and 7 days.
  • FIG. 4A shows images of the GNNS2 cells transfected with Ires or Ires-Cx43. The cells were stained with DAPI fluorescent dye (which stains all nuclei) and with the antibody against K ⁇ -67, showing images of both stains in the same field. Our results show that 2 days after transfection there are fewer K ⁇ -67 positive nuclei in the cells that we have restored Cx43. After 5 days no differences are observed.
  • Figure 4B shows the percentage of positive K ⁇ -67 nuclei with respect to the total number of nuclei, 2 and 5 days after the restoration of Cx43.
  • a significant decrease in the percentage of positive K ⁇ -67 nuclei is observed, 2 days after Transfection with the Ires-Cx43 vector compared to cells transfected with the empty vector. Five days after the transfection, no significant differences are observed. Therefore, the restoration of Cx43 in glioma stem cells reduces c-Src activity and proliferation rate. Since c-Src is an important regulator of stem cell self-renewal, it could be suggested that inhibition of proliferation is a consequence of inhibition of c-Src activity caused by Cx43.
  • Figure 5D shows that in GNNS2 cells expressing Cx43 the expression of N-cadherin decreases, suggesting that Cx43 promotes the change in the expression of cadherin isoforms in glioma stem cells. That is, there is a change of E-cadherin for N-cadherin.
  • Both the expression of Sox2 and the change from N-cadherin to E-cadherin are regulated by the Idl differentiation inhibitor. This protein binds to the transcription factors of type bHLH involved in cell differentiation and prevents their interaction with DNA.
  • the transcriptional regulator Id1 plays a critical role in modulating glioma cell invasiveness and the expression of mesenchymal markers.
  • FIG. 6A shows the immunocytochemistry of Id1, showing that levels of Id 1 decrease in cells expressing Cx43 compared to cells transfected with the plasmid Ires. More specifically, Id1 decreases in cells containing higher levels of Cx43 ( Figure 6B). The Western blot of Figure 6C confirmed the decrease in Id1 in the cells in which Cx43 has been restored.
  • Id1 is regulated by the activity of c-Src in different types of tumor cells, although there is nothing described of this relationship in gliomas.
  • Cx43 decreases the activity of c-Src, the expression of Id1, Sox2 and the change of expression of caderins. Therefore, the next objective was to determine if c-Src was the mediator in the effect of Cx43 on the glioma stem cell phenotype. To do this, the effect of c-Src inhibition on the expression of Id1 in glioma stem cells was first analyzed.
  • GNNS2 cells were cultured in the presence of the indicated concentrations of c-Src activity inhibitors: dasatinib, saracatinib and PP2. It was always used as a DMSO control, which is the vehicle used to dissolve these inhibitors. The cells were processed for Western blot analysis after 24 and 48 hours.
  • Figure 7A shows how the inhibition of c-Src activity with dasatinib decreases the levels of Id1 expression with respect to the control, at the two concentrations and at both 24 and 48 hours. These results were corroborated with saracatinib and PP2 ( Figure 7B).
  • Id1 regulates the expression of Sox2 and promotes the exchange of N-cadherin for E-cadherin, contributing to the reduction of the mesenchymal phenotype. Therefore, we study the effect of the inhibition of c-Src activity on the expression of Sox2 and the change of cadherins. For this, the activity of c-Src was inhibited with dasatinib using DMSO as control.
  • Figure 8A shows how inhibiting the activity of c-Src decreased the expression of Sox2 and N-cadherin with the consequent increase in E-cadherin (figure 8B). Therefore, the inhibition of c-Src exerts the same effect as the restoration of Cx43 on glioma stem cells.
  • the TAT-Cx43Src peptide [SEQ ID NO: 6] includes the c-Src binding domain and the two tyrosines phosphorylated by this kinase ( Figure 9A), fused to the penetrating TAT sequence [YGRKKRRQRRR (SEQ ID NO : 4)].
  • the other TAT-Cx43S peptide [SEQ ID NO: 5] includes the c-Src binding domain ( Figure 10A) fused to the penetrating TAT sequence [YGRKKRRQRRR (SEQ ID NO: 4)].
  • GNNS2 cells were incubated with the TAT-Cx43Src peptide, TAT-Cx43S or with the TAT peptide, used as a control, for 24 or 48 hours.
  • a immunocytochemistry of Cx43 is shown in Figure 9B, 24 hours after incubation with the peptide.
  • the TAT-Cx43Src peptide internalizes very effectively in all cells of the culture.
  • cells incubated with the TAT peptide show no expression of Cx43.
  • Western blot shows that the TAT-Cx43Src peptide temporarily decreased Src activity in glioma stem cells.
  • TAT-Cx43Src decreased the expression of Id1, N-cadherin (only at 48 hours) and Sox2 while E-cadherin expression increased compared to the TAT control peptide ( Figure 9C).
  • the TAT-Cx43S peptide also decreased activity of Src (figures 10B and 10C) and reversed the phenotype of glioma stem cells, since it reduced the expression of Id1, Sox2 and N-caderin while E-cadherin expression increased compared to the TAT control peptide (figure 10B ).
  • TAT-Cx43S penetrating peptide to reduce the expression of glioma stem cell phenotype determining proteins, inhibit proliferation, neurosphere formation and induce differentiation of glioma stem cells.
  • Neurosphere formation capacity was determined following the method described by Thirant et al. (Thirant et al., 201 1. PloS one, 6: e16375). 1 or 2 GNNS2 glioma stem cells were seeded per well in 96-well plates in RHB-A culture medium supplemented with 1% N2, 2% B27, 20 ng / ml EGF and 20 ng / ml b-FGF, in the presence or absence of the different penetrating peptides. After 4 weeks the number of neurospheres formed was counted.
  • the usual laboratory method was used for astrocyte cultures (Tabernero A. et al., 1996. Neurosci Res, 26: 369-376). To do this, brains of rats of 0 to 24 hours of life were used. All the steps of the process are performed under sterile conditions inside a laminar flow hood. After dissection of the animal, the tissue was removed and cleaned, removing the meninges and blood vessels. The material obtained was placed on a Petri dish with solution "A" containing: 0.3% bovine serum albumin and 40 pg / ml bovine pancreas DNase in saline. The brain was finely chopped in PBS with a scalpel and centrifuged.
  • the resulting precipitate was resuspended in the above solution, to which 0.025% trypsin of bovine pancreas was added, and incubated at 37 ° C, with stirring, for 15 min.
  • the trypsinization process was stopped by adding an equal volume of complete culture medium, consisting of Dubelcco's Modified Eagle Medium (DMEM), 10% fetal calf serum (FCS) and 40 pg / ml gentamicin.
  • DMEM Dubelcco's Modified Eagle Medium
  • FCS fetal calf serum
  • FCS fetal calf serum
  • Neuron cultures were performed according to the method described in our laboratory (Tabernero et al., 1993. Biochem J, 294: 635-638). Rat fetuses of 17.5 days of gestational age were used. The animals were obtained by rapid hysterectomy, after cervical dislocation of the mother. They were cleaned quickly and the umbilical cord was cut. Obtained the brains of these animals, the rest of the process was performed in the same way as the astrocyte culture. Under these conditions, cultures of 95% purity neurons are obtained, judging by their reaction with the specific antibody against the neurofilament.
  • New peptides were designed based on the region of Cx43 that interacts with c-Src ( Figure 12).
  • the first one comprises the consensus sequence of the C343 domain binding SH3, amino acids 274-283 [SEQ ID NO: 21 (PTAPLSPMSP)]
  • the second comprises the amino acid sequence 268-283 of the Cx43 [SEQ ID NO : 23 (FNGCSSPTAPLSPMSP)]
  • the third comprises the amino acid sequence 264-283 of Cx43 [SEQ ID NO: 25 (KYAYFNGCSSPTAPLSPMSP)].
  • TAT sequence of cellular internalization SEQ ID NO: 4 [YGRKKRRQRRR].
  • the final result was three internalizing sequences based on the binding region of Cx43 with c-Src.
  • SEQ ID NO: 22 - YGRKKRRQRRRPTAPLSPMSP penetrating peptide TAT-SH3
  • TAT-Cx43S (SEQ ID NO: 5), in addition to reversing the phenotype of GNNS2 glioma stem cells (figure 10), reduces the proliferation of G166 stem cells obtained from a glioblastoma of another patient (figure 1 one ).
  • Figure 1 1A shows that from concentrations of 25 ⁇ the TAT-Cx43S peptide reduces approximately 70% the cell growth rate compared to the TAT control peptide, whose growth rate was very similar to untreated cells.
  • concentrations of 50 and 75 ⁇ In addition, the reduction of cell growth is observed from 24 hours ( Figure 1 1 B), becoming more apparent after 48 and 72 hours.
  • the penetrating peptide TAT-Cx43S (SEQ ID NO: 5), in addition to reversing the phenotype of GNNS2 glioma stem cells (Figure 10), reduces the proliferation of G166 stem cells obtained from a glioblastoma of another patient.
  • FIG 14 shows that the MTT absorbance of penetrating peptide 268-283 (SEQ ID NO: 24) is significantly higher than that of penetrating peptide TAT-Cx43S (amino acids 266-283 of Cx43) (SEQ ID NO: 5). These results indicate that the penetrating peptide 268-283 (SEQ ID NO: 24) has less antitumor effect than TAT-Cx43S, which suggests that tyrosine 267 present in the penetrating peptide TAT-Cx43S influences the antitumor effect of this compound.
  • a peptide was designed that included TAT-Cx43S and one of the Cx43 tyrosines that phosphorylates c-Src, tyrosine 265.
  • a peptide comprising amino acids 264-283 of Cx43 (SEQ ID NO: 25) fused to the TAT penetrating sequence ( Figure 12), resulting in penetrating peptide 264-283 (SEQ ID NO: 26).
  • This penetrating peptide (SEQ ID NO: 26) has an antiproliferative effect very similar to TAT-Cx43S ( Figure 14).
  • TAT-Cx43Src and TAT-Cx43S reverse the stem cell phenotype.
  • TAT-Cx43Src and TAT-Cx43S reduce the expression of proteins responsible for the stem cell phenotype such as Sox-2 or Id1 and decrease proliferation and self-renewal capacity.
  • FIGS. 15 B and C show that TAT-Cx43Src and TAT-Cx43S, but not TAT-SH3 (SEQ ID NO: 22), increase the expression of the O4 differentiation marker.
  • TAT-Cx43Src and TAT-Cx43S reduce the capacity for self-renewal and promote the differentiation of glioma stem cells. Effect of penetrating peptides based on the connexin region43 that interacts with c-Src in different cell types.
  • FIG. 17 B shows that in the control situation or in the presence of the TAT sequence, glioma stem cells are characterized by the presence of human nestin on the set of astrocytes (nuclei without nestin).
  • TAT-Cx43S human nestin staining is greatly reduced, with most of the astrocyte cells judging by nuclear staining.
  • TAT-Cx43S selectively affects glioma stem cells without modifying the viability of quiescent and differentiated cells such as astrocytes and neurons.

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Abstract

La présente invention concerne des peptides qui comprennent le fragment 266-283 de la connexine 43 de SEQ ID n°1 et l'utilisation de ces derniers pour le traitement de maladies qui vont de pair avec la prolifération cellulaire, en particulier pour le traitement de gliomes. L'invention porte également sur la ou les compositions pharmaceutiques et la trousse qui comprend lesdits peptides.
PCT/ES2014/070444 2013-05-30 2014-05-30 Peptides dérivés de la connexine 43 et composition pharmaceutique pour le traitement du cancer WO2014191608A1 (fr)

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ES201330793A ES2526109B1 (es) 2013-05-30 2013-05-30 Péptido y composición farmacéutica para el tratamiento del cáncer

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190148281A1 (en) * 2017-11-15 2019-05-16 Sharp Kabushiki Kaisha Power semiconductor module and electronic device
WO2021021716A1 (fr) * 2019-07-26 2021-02-04 University Of Virginia Patent Foundation Compositions et procédés pour inhiber la formation d'une néointima
EP4209217A1 (fr) * 2022-01-11 2023-07-12 Fundación Profesor Novoa Santos Procédé de traitement d'un cancer à brca1/2 muté
ES2946554A1 (es) * 2022-01-19 2023-07-20 Univ Salamanca Peptidos y composicion farmaceutica para el tratamiento de tumores

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WO1999010376A1 (fr) * 1997-08-22 1999-03-04 Washington University Systeme regulateur inductible et son utilisation

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WO1999010376A1 (fr) * 1997-08-22 1999-03-04 Washington University Systeme regulateur inductible et son utilisation

Non-Patent Citations (6)

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Title
DATABASE EMBL/GENBANK/DDBJ August 2000 (2000-08-01), SCHOENFELDER, M. ET AL.: "Expression of connexins in bovine oviduct''.", accession no. 9GJX4 *
DATABASE EMBL/GENBANK/DDBJ August 2009 (2009-08-01), MEIXNER, M.: "Gap junction protein alpha 1. Felis catus (Felis silvestris catus)''.", accession no. OX650 *
GANGOSO, E. ET AL.: "A cell -penetrating peptide based on the interaction between c-Src and Connexin43 reverses glioma stem cell phenotype''.", CELL DEATH AND DISEASES., vol. 5, 30 January 2014 (2014-01-30), pages 1 - 13 *
HERRERO-GONZÁLEZ, S. ET AL.: "Connexin43 inhibits the oncogenic activity of c-Scr in C6 glioma cells''.", ONCOGENE, vol. 29, no. 42, 21 October 2010 (2010-10-21), pages 5712 - 5723 *
SORGEN, P.L. ET AL.: "Structural changes in the carboxyl terminus of the gap junction protein Connexin 43 indicates signaling between binding domains for c-Src and zonula occludens-1''.", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, no. 52, 24 December 2004 (2004-12-24), pages 54695 - 54701, XP009107524, DOI: doi:10.1074/jbc.M409552200 *
YU , S-C. ET AL.: "Connexin 43 reverses malignant phenotypes of glioma stem cells by modulating E-Cadherin''.", STEM CELLS., vol. 30, no. 2, February 2012 (2012-02-01), pages 108 - 120 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190148281A1 (en) * 2017-11-15 2019-05-16 Sharp Kabushiki Kaisha Power semiconductor module and electronic device
WO2021021716A1 (fr) * 2019-07-26 2021-02-04 University Of Virginia Patent Foundation Compositions et procédés pour inhiber la formation d'une néointima
EP4209217A1 (fr) * 2022-01-11 2023-07-12 Fundación Profesor Novoa Santos Procédé de traitement d'un cancer à brca1/2 muté
WO2023135169A1 (fr) * 2022-01-11 2023-07-20 Fundación Profesor Novoa Santos Procédé de traitement du cancer à mutation de brca1/2
ES2946554A1 (es) * 2022-01-19 2023-07-20 Univ Salamanca Peptidos y composicion farmaceutica para el tratamiento de tumores
WO2023139156A1 (fr) 2022-01-19 2023-07-27 Universidad De Salamanca Peptides et composition pharmaceutique pour le traitement de tumeurs

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ES2526109A2 (es) 2015-01-05
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