Patent Grant
8450279
The present invention relates to cell permeable Nm23 recombinant proteins in which a metastasis suppressor Nm23 is fused to a macromolecule transduction domain (MTD), polynucleotides encoding the same, expression vectors for producing the same, and anti-metastatic pharmaceutical compositions including the same as effective ingredients for inhibiting metastasis.
Nm23 gene has been reported to encode proteins involved in the development and differentiation of a normal tissue, and its expression is decreased in various metastatic cells. Nm23 proteins belong to a tumor metastasis suppressor and generally consist of 150 to 180 amino acids. Nm23 proteins contain a leucine zipper motif and exhibit nucleoside diphosphate kinase (NDPK) activity. Two human Nm23 homologues, Nm23-H1 and Nm23-H2, consist of 152 amino acids having molecular weights of 17,143 and 17,294, respectively. In particular, it has been found that Nm23-H1 plays an important role in tumor metastasis and other various cell mechanisms including cell proliferation, embryogenesis, differentiation and tumorigenesis.
The mechanisms by which Nm23 affects tumor development and metastasis have not yet been clearly investigated. NDPK transfers a phosphoryl group between nucleoside triphosphate and nucleoside diphosphate via a covalent phosphoenzyme intermediate. For such a phosphorylation, histidine 118 of each Nm23-H1 and Nm23-H2 is served as a target site. Apart from the NDPK-mediated histidine phosphorylation, serine autophosphorylation was observed in Nm23 (MacDonald N J et al., J. Biol. Chem. 268: 25780-25789, 1993). When melanoma cells of Nm23 transfected mice were compared with a control cell, there was a direct correlation between the in vivo phosphorylation level of Nm23 serine and the inhibition of tumor metastatic potential. Serine phosphorylation of mouse Nm23 is inhibited by cAMP in vivo, while by forskolin in vitro, which demonstrates that the phosphorylation is controlled by signal transduction pathways.
Initially, Nm23 expression was reported to closely correlate to mouse melanoma cell lines with poor metastatic potential. The relationship between Nm23 reduced expression and tumor metastasis has been regarded as direct evidence to support the fact that Nm23 functions as a tumor metastasis suppressor (Steeg, P. S., Breast Dis. 10: 47-50, 1998). Inducible overexpression of Nm23 exhibited remarkably reduced metastatic potential in a highly metastatic cancer cell line. The Nm23 gene cloned as a putative tumor metastasis suppressor gene exhibits serine/threonine specific phosphotransferase and histidine protein kinase activities, as well as NDPK activity. Further, the expression of Nm23 is reduced as hematopoietic stem cells (HSC) are differentiated, which suggests that Nm23 is an important factor for anti-differentiation in those cells (Gervasi, F. et al., Cell Growth Differ 7: 1689-95, 1996). It has been found that Nm23 exhibits a strong inhibitory effect on tumor metastasis upon temporary transfection in forced inducible gene expression and in vitro metastasis model systems of human tumors (Hirayama, R. et al., J. Natl. Cancer Inst. 83: 1249-50, 1991; Nakayama, T. et al., J. Natl. Cancer Inst. 84: 1349-54, 1992; Leone, A. et al., Oncogene 8: 2325-33, 1993; Leone, A. et al., Oncogene 8: 855-65, 1993). In contrast, Nm23 mutation, leading to the loss of NDPK activity, had no influence on the inhibitory function of Nm23 in breast cancer cells (MacDonald, N. J. et al., J. Biol. Chem. 271:25107-16, 1996).
The most authentic evidence for the fact that Nm23 is a metastasis suppressor is revealed when the Nm23 gene is transfected into tumor cell lines. In metastatically competent cells, the administration of high dose Nm23 showed reduced metastatic activity by 40 to 98% as compared with a control transfectant (Leone, A. et al, Cell 65: 25-35, 1991; Leone, A. et al., Oncogene 8:2325-33, 1993).
Recently, it has been reported that Nm23 interacts with a kinase suppressor of Ras (KSR) discovered in the Drosophilae (Drosophilar melanogaster) and nematode (Caenorhabditis elegans) systems (Morrison, D. K., J. Cell Sci. 114: 1609-12, 2001). KSR is a scaffold protein of a mitogen-activated protein kinase (MAPK) cascade (Burack, W. R. and Shaw, A. S., Curr. Opin. Cell Biol. 12: 211-6, 2000; Pawson, T. and Scott, J. D., Science 278: 2075-80, 1997). Such scaffold protein is necessary to enhance the rate of phosphorylation and contribute to the specificity and stabilization of the phosphorylation pathway. Once the MAPK signal transduction pathway is activated by active Ras, KSR is forcedly dephosphorylated, and then, serve as a scaffold for the activation of MAPK cascade. During this process, Nm23 phosphorylates KSR serine 392, which is a binding site for another associated protein of KSR. If the serine 392 is mutated, Nm23 phosphorylates KSR serine 434. The metastatic inhibitory activity of Nm23 has been clearly demonstrated by the fact that metastatic potential is inhibited in various tumor cells transfected with Nm23 gene (Yoshida, T. et al., J Gastroenterol. 35: 768-74, 2000). In cells activated by the stimulation of the MAPK cascade, the interaction between Nm23 and KSR induces KSR phosphorylation in vitro in a complicated manner via a histidine-dependent pathway (Hartsough, M. T. et al., J. Biol. Chem. 277: 32389-99, 2002). Further, the in vivo association of KSR and Nm23 inhibits the phenotypic effect of active Ras which activates the MAPK cascade.
Accordingly, the administration of high dose Nm23 protein may phosphorylate and inactivate KSR in vivo, leading to the inhibition of Ras-mediated MAPK cascade. The present inventors have therefore believed that the inhibition of MAPK signal transduction pathway mediated by re-phosphorylation of KSR may inhibit cell proliferation, differentiation and migration of cancer cells and exhibit anti-metastatic effect in various human cancers, and endeavored to develop new anti-metastatic agents by using the Nm23 protein.
Meanwhile, small molecules derived from synthetic compounds or natural compounds can be transported into the cells, whereas macromolecules, such as proteins, peptides, and nucleic acids, cannot. It is widely understood that macromolecules larger than 500 kDa are incapable of penetrating the plasma membrane, i.e., the lipid bilayer structure, of live cells. To overcome this problem, a macromolecule intracellular transduction technology (MITT) was developed (Jo et al., Nat. Biotech. 19: 929-33, 2001), which allows the delivery of therapeutically effective macromolecules into cells, making the development of new drugs using peptides, proteins and genetic materials possible. According to this method, if a target macromolecule is fused to a hydrophobic macromolecule transduction domain (MTD) and other cellular delivery regulators, synthesized, expressed, and purified in the form of a recombinant protein, it can penetrate the plasma membrane lipid bilayer of the cells, be accurately delivered to a target site, and then, effectively exhibit its therapeutic effect. Such MTDs facilitate the transport of many impermeable materials which are fused to peptides, proteins, DNA, RNA, synthetic compounds, and the like into the cells.
Accordingly, the inventors of the present invention have developed a method of mediating the transport of a metastasis suppressor Nm23 into the cells, where cell permeable Nm23 recombinant proteins are engineered by fusing a MTD to the metastasis suppressor Nm23. Such cell permeable Nm23 recombinant proteins have been found to efficiently mediate the transport of a metastasis suppressor Nm23 into the cells in vivo as well as in vitro and can be used as anti-metastatic agents for inhibiting metastasis occurring in various human cancers.
Accordingly, the objective of the present invention is to provide cell permeable Nm23 recombinant proteins as an anti-metastatic agent which is effective for preventing metastasis in various kinds of human cancers by inhibiting proliferation, differentiation and migration of cancer cells.
One aspect of the present invention relates to cell permeable Nm23 recombinant proteins capable of mediating the transport of a metastasis suppressor Nm23 into a cell by fusing a macromolecule transduction domain (MTD) having cell permeability to the metastasis suppressor protein.
Another aspect of the present invention relates to polynucleotides encoding the above cell permeable Nm23 recombinant proteins.
The present invention also relates to expression vectors containing the above polynucleotides, and transformants transformed with the above expression vectors.
Another aspect of the present invention relates to a method of producing cell permeable Nm23 recombinant proteins involving culturing the above transformants.
Another aspect of the present invention relates to a pharmaceutical composition including the above cell permeable Nm23 recombinant proteins as an effective ingredient for inhibiting metastasis.
a is a photograph of an agarose gel electrophoresis analysis showing PCR-amplified DNA fragments encoding cell permeable Nm23 recombinant proteins being fused to a kFGF4-derived MTD and constructed in the full-length forms according to the present invention.
b is a photograph of an agarose gel electrophoresis analysis showing PCR-amplified DNA fragments encoding cell permeable Nm23 recombinant proteins being fused to each of JO-76 and JO-77 MTDs and constructed in the full-length forms according to the present invention.
a is a schematic diagram illustrating the subcloning of a PCR product encoding a cell permeable Nm23 recombinant protein into the pGEM-T Easy vector according to the present invention.
b and 3c are photographs of an agarose gel electrophoresis analysis showing the PCR products encoding the cell permeable Nm23 recombinant proteins from
a is a schematic diagram illustrating the cloning of a recombinant DNA fragment encoding a cell permeable Nm23 recombinant protein into the pET 28(+) vector according to the present invention.
b and 4c are photographs of an agarose gel electrophoresis analysis showing the recombinant DNA fragments encoding the cell permeable Nm23 recombinant proteins subcloned in the pET 28(+) vector according to the present invention.
a and 7b are graphs illustrating the results of flow cytometry analysis of cell permeabilities of cell permeable Nm23 recombinant proteins according to the present invention.
a to 8c are confocal laser scanning microscopy photographs visualizing the cell permeabilities of cell permeable Nm23 recombinant proteins according to the present invention in mouse fibroblasts.
a and 9b are photographs of a Western blot analysis showing the inhibitory effect of cell permeable Nm23 recombinant proteins according to the present invention on MAPK signal transduction.
a and 10b are photographs of an invasion analysis showing the inhibitory effect of cell permeable Nm23 recombinant proteins according to the present invention on metastasis.
a is a photograph illustrating the inhibitory effect on metastasis in a mouse lung tissue extracted from a mouse administered with the cell permeable Nm23 recombinant protein according to the present invention.
b is a photograph of immunohistochemical staining showing the expression of a metastatic marker, vimentin, in a mouse lung tissue extracted from a mouse administered with the cell permeable Nm23 recombinant protein according to the present invention.
The present invention provides cell permeable Nm23 recombinant proteins (CP-Nm23) capable of mediating the transport of a metastasis suppressor Nm23 into a cell in which the metastasis suppressor Nm23 is fused to a macromolecule transduction domain and, thereby, imparted with cell permeability; and polynucleotides encoding each of the cell permeable Nm23 recombinant proteins.
The present invention is characterized in that a metastasis suppressor Nm23 which is a macromolecule incapable of being introduced into a cell is fused to a specific macromolecule transduction domain (hereinafter, “MTD”) peptide so as to provide cell permeability, and thus, can be effectively transported into a cell. The MTD peptide may be fused to the N-terminus, the C-terminus, or both termini of the metastasis suppressor Nm23.
The present invention relates to cell permeable Nm23 recombinant proteins that are engineered by fusing a metastasis suppressor Nm23 to one of three MTD domains capable of mediating the transport of a macromolecule into a cell.
The term “cell permeable Nm23 recombinant protein” as used herein refers to a covalent bond complex bearing a MTD and a metastasis suppressor protein Nm23, where they are functionally linked by genetic fusion or chemical coupling. Here, the term “genetic fusion” refers to a co-linear, covalent linkage of two or more proteins or fragments thereof via their individual peptide backbones, through genetic expression of a polynucleotide molecule encoding those proteins.
Nm23 is a metastasis suppressor protein which inhibits the proliferation, differentiation and migration of cancer cells and induces apoptosis by controlling the MAPK signal transduction cascade which is mediated by KSR phosphorylation. Nm23 has an amino acid sequence represented by SEQ ID NO: 2, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 1. Nm23 functions as an important target protein in signal transduction cascades including KSR and Ras-mediated MAPK.
It has been reported that Nm23 is an endogeneous protein and exhibits NDP (nucleotide diphosphate)-kinase enzyme activity (Biggs et al., Cell 63, 933-940, 1990). Nm23 has also been found to be a transcription factor and cell differentiation inhibitor (I factor) (Postel et al., Science 261, 478-480, 1993; Okabe-Kado et al., Biochim. Biophys. Acta. 1267, 101-106, 1995).
In humans, eight nm23 isotypes (nm23-H1, nm23-H2, DR-nm23, nm23-H4, nm23-H5, nm23-H6, nm23-H7, and nm23-H8) have been identified to date, all of which are implicated in the regulation of metastasis (Rosengard et al., Nature 342, 177-180, 1989; Charpin C. et al., Int. J. Cancer 74, 416-420, 1997). In certain embodiments, cell permeable recombinant proteins for Nm23-H1 have been constructed, but are not limited thereto.
For the MTD capable of being fused to the metastasis suppressor Nm23, cell permeable peptides having an amino acid sequence selected from the group consisting of SEQ ID NOS: 4, 6, 8, and 37 to 227 may be used. The MTD having one of the amino acid sequences represented by SEQ ID NOS: 4, 6, 8 and 37 to 227 is a cell permeable polypeptide which is capable of mediating the transport of a biologically active molecule, such as a polypeptide, a protein domain, or a full-length protein across the cell membrane. Suitable MTDs for the present invention include a hydrophobic region showing cell membrane targeting activity by forming a helix structure at a signal peptide which is composed of an N-terminal domain, a hydrophobic domain and a C-terminal domain containing a secreted protein cleavage site. These MTDs can directly penetrate the cell membrane without causing any cell damage, transport a target protein into a cell, and thus, allow the target protein to exhibit its desired function.
The MTDs having the amino acid sequences represented by SEQ ID NOS: 4, 6, 8, and 37 to 227 and capable of being fused to a metastasis suppressor Nm23 according to the present invention are summarized in the following Tables 1a to 11.
coelicolor A3(2)]
coelicolor A3(2)]
Enterica serovar Typhi]
Mus musculus
Typhi (Salmonella typhi) strain CT18
In some embodiments, the present invention may employ a kaposi fibroblast growth factor 4 (kFGF4)-derived MTD having the nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4 (hereinafter, “MTD1”), a JO-76 MTD having the nucleotide sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6 which is a hypothetical protein derived from Theileria annulata (hereinafter, “MTD2”), and a JO-77 MTD having the nucleotide sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8 which belongs to member B of a human TMEM9 domain family (hereinafter, “MTD3”), as the MTD capable of mediating the transport of the metastasis suppressor Nm23 into a cell.
The cell permeable Nm23 recombinant proteins according to the present invention have a structure where one of the three MTDs (kFGF4-derived MTD: MTD1, JO-76: MTD2, JO-77: MTD3) is fused to one terminus or both termini of a metastasis suppressor protein Nm23, and a SV40 large T antigen-derived nuclear localization sequence (NLS)(nucleotide sequence of SEQ ID NO: 9, amino acid sequence of SEQ ID NO:10) and a histidine-tag (His-Tag) affinity domain for easy purification are fused to one terminus of the resulting construct.
In another embodiment, the present invention relates to the construction of eight full-length forms of a cell permeable Nm23 recombinant protein by using one of a kFGF4-derived MTD, a JO-76 MTD and a JO-77 MTD.
As used herein, the term “full-length form” refers to a construct including the entire amino acid sequence of the metastasis suppressor protein Nm23 which does not contain any deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 2. However, it should be obvious to a skilled person in the art that Nm23 derivatives including various kinds of modifications through the deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 2 that is made within the scope of not causing a deterioration of the Nm23 anti-metastatic effect can be used in the present invention.
Referring to
1) His-MTD1-Nm23 (HM1N), where a kFGF4-derived MTD is fused to the N-terminus of a full-length Nm23,
2) His-Nm23-MTD1 (HNM1), where a kFGF4-derived MTD is fused to the C-terminus of a full-length Nm23,
3) His-MTD1-Nm23-MTD1 (HM1NM1) where a kFGF4-derived MTD is fused to both termini of a full-length Nm23,
4) His-MTD2-Nm23 (HM2N), where a JO-76 MTD is fused to the N-terminus of a full-length Nm23,
5) His-Nm23-MTD2 (HNM2), where a JO-76 MTD is fused to the C-terminus of a full-length Nm23,
6) His-MTD3-Nm23 (HM3N), where a JO-77 MTD is fused to the N-terminus of a full-length Nm23,
7) His-Nm23-MTD3 (HNM3), where a JO-77 MTD is fused to the C-terminus of a full-length Nm23, and
8) His-MTD3-Nm23-MTD3 (HM3NM3), where a JO-77 MTD is fused to both termini of a full-length Nm23,
where a His-tag and a NLS derived from SV40 large T antigen are covalently coupled to the N-terminus of the above constructs.
As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a kFGF4-derived MTD as described above, His-MTD1-Nm23 (HM1N) has an amino acid sequence represented by SEQ ID NO: 22, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 21; His-Nm23-MTD1 (HNM1) has an amino acid sequence represented by SEQ ID NO: 24, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 23; and His-MTD1-Nm23-MTD1 (HM1NM1) has an amino acid sequence represented by SEQ ID NO: 26, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 25.
As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a JO-76 MTD as described above, His-MTD2-Nm23 (HM2N) has an amino acid sequence represented by SEQ ID NO: 28, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 27; and His-Nm23-MTD2 (HNM2) has an amino acid sequence represented by SEQ ID NO: 30, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 29.
As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a JO-77 MTD as described above, His-MTD3-Nm23 (HM3N) has an amino acid sequence represented by SEQ ID NO: 32, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 31; His-Nm23-MTD3 (HNM3) has an amino acid sequence represented by SEQ ID NO: 34, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 33; and His-MTD3-Nm23-MTD3 (HM3NM3) has an amino acid sequence represented by SEQ ID NO: 36, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 35.
As a control for the cell permeable Nm23 recombinant proteins, His-Nm23 (HN), where a full-length Nm23 is fused only to a NLS derived from SV40 large T antigen and a histidine-tag (His-Tag) without any MTD, is constructed. The control protein has an amino acid sequence represented by SEQ ID NO: 20, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 19.
Further, the present invention provides an expression vector containing the polynucleotide encoding each of the cell permeable Nm23 recombinant proteins described above, and a transformant capable of producing each of the cell permeable Nm23 recombinant proteins at high levels, which is obtainable by transforming a host cell using the expression vector.
As used herein, the term “expression vector” is a vector capable of expressing a target protein or a target RNA in a suitable host cell. The nucleotide sequence of the present invention may be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the nucleotide sequence by a suitable host cell.
Within an expression vector, the term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence. The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements. Such operable linkage with the expression vector can be achieved by conventional gene recombination techniques known in the art, while site-directed DNA cleavage and linkage are carried out by using conventional enzymes known in the art.
The expression vectors suitable for the present invention may include plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors and the like, but are not limited thereto. The expression vectors for use in the present invention may contain a signal sequence or a leader sequence for membrane targeting or secretion, as well as regulatory sequences such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, an enhancer and the like. The promoter may be a constitutive or an inducible promoter. Further, the expression vector may include one or more selectable marker genes for selecting the host cell containing the expression vector, and may further include a nucleotide sequence that enables the vector to replicate in the host cell in question.
The expression vector constructed according to the present invention may be exemplified by pET28a(+)-HNM1 where the polynucleotide encoding the recombinant protein HNM1 where a kFGF4-derived MTD is fused to the C-terminus of a full-length Nm23 is inserted into a cleavage site of NdeI restriction enzyme within the multiple cloning sites (MCS) of a pET-28a(+) vector.
In another embodiment, the polynucleotide of the present invention is cloned into a pET-28a(+) vector (NOVAGEN, USA) bearing a His-tag sequence so as to fuse six histidine residues to the N-terminus of the cell permeable Nm23 recombinant protein to allow easy purification.
Accordingly, the cell permeable Nm23 recombinant protein expressed in the above expression vector has a structure where one of a kFGF4-derived MTD, a JO-76 MTD and a JO-77 MTD is fused to the full-length or truncated Nm23, and a His-tag and NLS are linked to the N-terminus thereof.
The present invention further provides a transformant capable of producing each of the cell permeable Nm23 recombinant proteins at high levels which is obtainable by transforming a host cell using the expression vector. The host cell suitable for the present invention may be eukaryotic cells, such as E. coli. In one embodiment of the present invention, E. coli used as a host cell is transformed with the expression vector, for example, pET28a(+)-HNM1 containing the polynucleotide encoding the cell permeable recombinant protein HNM1 where a kFGF4-derived MTD is fused to the C-terminus of a full-length Nm23 according to the present invention so as to produce the cell permeable Nm23 recombinant protein at high levels. Methods for transforming bacterial cells are well known in the art, and include, but are not limited to, biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, microprojectile bombardment, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, PEG-mediated fusion and liposome-mediated method.
In some embodiments, the transformants obtained by transforming E. coli DH5α with the expression vector containing the cell permeable Nm23 recombinant protein HM3N where a JO-77 MTD is fused to the N-terminus of a full-length Nm23, and the expression vector containing the cell permeable Nm23 recombinant protein HNM3 where a JO-77 MTD is fused to the C-terminus thereof, respectively, were deposited under accession numbers KCTC-11380BP and KCTC-11381BP, respectively, with the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52, Oun-Dong, Yusong-Ku, Taejon 305-333, Republic of Korea. All deposits referred to herein were made on Aug. 28, 2008 in accordance with the Budapest Treaty, and all restrictions imposed by the depositor on the availability to the public of the deposited biological material will be irrevocably removed upon the granting of the patent.
The present invention provides a method of producing the cell permeable Nm23 recombinant proteins at high levels, which includes the step of culturing the above transformant.
The method of the present invention may be carried out by culturing the transformant in a suitable medium under suitable conditions for expressing a cell permeable Nm23 recombinant protein of the present invention in the expression vector introduced into the transformant. Methods for expressing a recombinant protein by culturing a transformant are well known in the art, and for example, may be carried out by inoculating a transformant in a suitable medium for growing the transformant, performing a subculture, transferring the same to a main culture medium, culturing under suitable conditions, for example, supplemented with a gene expression inducer, isopropyl-J3-D-thiogalactoside (IPTG) and, thereby, inducing the expression of a recombinant protein. After the culture is completed, it is possible to recover a “substantially pure” recombinant protein from the culture solution. The term “substantially pure” means that the recombinant protein and polynucleotide encoding the same of the present invention are essentially free of other substances with which they may be found in nature or in vivo systems to the extent practical and appropriate for their intended use.
A recombinant protein of the present invention obtained as above may be isolated from the inside or outside (e.g., medium) of host cells, and purified as a substantially pure homogeneous polypeptide. The method for polypeptide isolation and purification is not limited to any specific method. In fact, any standard method may be used. For instance, chromatography, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, and recrystallization may be appropriately selected and combined to isolate and purify the polypeptide. As for chromatography, affinity chromatography, ion-exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography, etc., for example, may be used (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989; Deutscher, M., Guide to Protein Purification Methods Enzymology vol. 182. Academic Press. Inc., San Diego, Calif., 1990).
Meanwhile, the recombinant protein expressed in the transformants according to the present invention can be classified into a soluble fraction and an insoluble fraction according to protein characteristics during the protein purification process. If the majority of the expressed recombinant proteins are present in the soluble fraction, the recombinant protein can be isolated and purified according to the method as described above. However, when the majority of the expressed recombinant proteins are present in the insoluble fraction, i.e., as inclusion bodies, the recombinant proteins are first solubilized by using polypeptide denaturing agents, e.g., urea, guanidine HCl, or detergents, and then, purified by performing a series of centrifugation, dialysis, electrophoresis and column chromatography. Since there is the risk of losing the recombinant protein's activity due to a structural modification caused by the polypeptide denaturing agent, the process of purifying the recombinant protein from the insoluble fraction requires desalting and refolding steps. That is, the desalting and refolding steps can be performed by dialysis and dilution with a solution that does not include a polypeptide denaturing agent or by centrifugation with a filter. Further, if a salt concentration of the solution used for the purification of a recombinant protein from a soluble fraction is relatively high, such desalting and refolding steps may be performed.
In some embodiments, it has been found that the cell permeable Nm23 recombinant protein of the present invention mostly exists in the insoluble fraction as an inclusion body. In order to purify the recombinant protein from the insoluble fraction, the insoluble fraction may be dissolved in a lysis buffer containing a non-ionic surfactant such as Triton X-100, subjected to ultrasonification, and then centrifuged to separate a precipitate. The separated precipitate may be dissolved in a buffer supplemented with a strong denaturing agent, such as urea, and centrifuged to separate the supernatant. The above separated supernatant is purified by means of a histidin-tagged protein purification kit and subjected to ultrafiltration, for example, by using an amicon filter for salt removal and protein refolding, thereby obtaining a purified recombinant protein of the present invention.
Further, the present invention provides an anti-metastatic pharmaceutical composition including the cell permeable Nm23 recombinant protein as an effective ingredient for preventing metastasis by inhibiting the proliferation, differentiation and migration of cancer cells and inducing apoptosis.
The metastasis suppressor Nm23, which functions as an important target protein for signal transduction cascades including KSR and Ras-mediated MAPK, can control the MAPK signal transduction cascade mediated by KSR phosphorylation, and thus, inhibit the proliferation, differentiation and migration of cancer cells and induce apoptosis. Therefore, the cell permeable Nm23 recombinant proteins of the present invention can be effectively used as an anti-metastatic agent capable of preventing and/or treating cancer metastasis.
The cell permeable Nm23 recombinant proteins of the present invention can activate cell signaling mechanisms involved in the activation of ATM and p53 that induce cell cycle arrest and apoptosis in response to DNA damage or oncogenic signals by efficiently introducing a metastasis suppressor protein Nm23 into a cell. Therefore, the cell permeable Nm23 recombinant proteins of the present invention can be effectively used as an anti-metastatic agent for treating various kinds of human cancers.
The pharmaceutical composition comprising the recombinant protein of the present invention as an effective ingredient may further include pharmaceutically acceptable carriers suitable for oral administration or parenteral administration. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995). The carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. In case of oral administration, the recombinant protein of the present invention can be formulated in the form of chewable tablets, buccal tablets, troches, capsules, elixir, suspensions, syrup, wafers or combination thereof by mixing with the carriers. Further, the carriers for parenteral administration may include water, suitable oil, saline, aqueous glucose, glycol and the like, and may further include stabilizers and preservatives. The stabilizers suitable for the present invention may include antioxidants such as sodium bisulfite, sodium sulfite and ascorbic acid. Suitable preservatives may include benzalconium chloride, methly-paraben, propyl-paraben and chlorobutanol.
The pharmaceutical composition of the present invention may be formulated into various parenteral or oral administration forms. Representative examples of the parenteral formulation include those designed for administration by injection. For injection, the recombinant proteins of the present invention may be formulated in aqueous solutions, specifically in physiologically compatible buffers or physiological saline buffer. These injection formulations may be formulated by conventional methods using one or more dispersing agents, wetting agents and suspending agents. For oral administration, the proteins can be readily formulated by combining the proteins with pharmaceutically acceptable carriers well known in the art. Such carriers enable the proteins of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Such oral solid formulations may include suitable excipients such as diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol cellulose and/or glycin) and lubricants (e.g., colloidal silica, talc, stearic acid, magnesium stearate, calcium stearate, and/or polyethylene glycol). The tablets may include binders, such as aluminum silicate, starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP), and disintegrating agents, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, absorbents, coloring agents, flavoring agents and/or sweeteners may be added. The formulations can be prepared by mixing, granulating or coating according to conventional methods well-known in the art.
If necessary, the pharmaceutical compositions of the present invention may further include pharmaceutical additives, such as preservatives, antioxidants, emulsifiers, buffering agents and/or salts for regulating osmosis and other therapeutically effective materials, and can be formulated according to conventional methods known in the art.
In addition, the pharmaceutical composition of the present invention can be administered via oral routes or parenteral routes such as intravenously, subcutaneously, intranasally or intraperitoneally. The oral administration may include sublingual application. The parenteral administration may include drip infusion and injection such as subcutaneous injection, intramuscular injection, intravenous injection and introtumoral injection.
The total effective amount of the recombinant protein of the present invention can be administered to patients in a single dose or can be administered by a fractionated treatment protocol, in which multiple doses are administered over a more prolonged period of time. Although the amount of the recombinant protein or nucleic acid encoding the same in the pharmaceutical composition of the present invention may vary depending on the severity of diseases, the protein or the nucleic acid may be generally administered several times a day at an effective dose of 5 to 20 mg. However, a suitable dose of the recombinant protein in the pharmaceutical composition of the present invention may depend on many factors, such as age, body weight, health condition, sex, disease severity, diet and excretion of patients, as well as the route of administration and the number of treatments to be administered. In view of the above factors, any person skilled in the art may determine the effective dose of the recombinant protein as an anti-metastatic agent for preventing metastasis in various human cancers. The pharmaceutical composition of the present invention containing the recombinant protein has no special limitations on its formulation, administration route and/or administration mode insofar as it exhibits the effects of the present invention.
The following examples are provided to illustrate the embodiments of the present invention in more detail, but are by no means intended to limit its scope.
Eight full-length forms of a cell permeable Nm23 (CP-Nm23) recombinant protein were constructed by using one of a kFGF4-derived MTD (MTD1), a JO-76MTD (MTD3) and a JO-77 MTD (MTD2) as a macromolecule transduction domain.
Referring to
1) His-MTD1-Nm23 (HM1N), where a kFGF4-derived MTD is fused to the N-terminus of a full-length Nm23,
2) His-Nm23-MTD1 (HNM1), where a kFGF4-derived MTD is fused to the C-terminus of a full-length Nm23,
3) His-MTD1-Nm23-MTD1 (HM1NM1) where a kFGF4-derived MTD is fused to both termini of a full-length Nm23,
4) His-MTD2-Nm23 (HM2N), where a JO-76 MTD is fused to the N-terminus of a full-length Nm23,
5) His-Nm23-MTD2 (HNM2), where a JO-76 MTD is fused to the C-terminus of a full-length Nm23,
6) His-MTD3-Nm23 (HM3N), where a JO-77 MTD is fused to the N-terminus of a full-length Nm23,
7) His-Nm23-MTD3 (HNM3), where a JO-77 MTD is fused to the C-terminus of a full-length Nm23, and
8) His-MTD3-Nm23-MTD3 (HM3NM3), where a JO-77 MTD is fused to both termini of a full-length Nm23,
where a His-tag and a NLS derived from SV40 large T antigen are covalently coupled to the N-terminus of the above constructs.
In order to prepare the full-length CP-Nm23 recombinant constructs, polymerase chain reactions (PCRs) were carried out by using the oligonucleotides described in Table 1 below as a primer pair specific for each recombinant construct and a human Nm23 cDNA (SEQ ID NO: 1) as a template. The forward and reverse primers for amplifying HM1N have nucleotide sequences represented by SEQ ID NOS: 13 and 12, respectively; those for amplifying HNM1 have nucleotide sequences represented by SEQ ID NOS: 11 and 14, respectively; those for amplifying HM1NM1 have nucleotide sequences represented by SEQ ID NOS: 13 and 14, respectively; those for amplifying HM2N have nucleotide sequences represented by SEQ ID NOS: 15 and 12, respectively; those for amplifying HNM2 have nucleotide sequences represented by SEQ ID NOS: 11 and 16, respectively; those for amplifying HM3N have nucleotide sequences represented by SEQ ID NOS: 17 and 12, respectively; those for amplifying HNM3 have nucleotide sequences represented by SEQ ID NOS: 11 and 18, respectively; and those for amplifying HM3NM3 have nucleotide sequences represented by SEQ ID NOS: 17 and 18, respectively.
The oligonucleotides as a forward and reverse primer set specific for each recombinant protein are summarized in Table 2 below.
The PCR was performed in a 50 μl reaction mixture containing 100 ng of human Nm23 cDNA as a template, 0.2 mM dNTP mixture (dGTP, dATP, dTTP, and dCTP, each at 2 mM), 0.6 μM of each primer, 5 of 10×Taq buffer, 1 μl of Taq polymerase (TAKARA, Japan). The PCR was performed for 25 cycles at 94° C. for 45 seconds, at 53° C. for 45 seconds and at 72° C. for 45 seconds after the initial denaturation of 94° C. for 2 minutes, followed by the final extension at 72° C. for 5 minutes. After the PCR was completed, the amplified PCR product was digested with restriction enzyme Ndel and loaded onto a 1.0% agarose gel and fractionated. As shown in
The DNA band of expected size was excised from the gel, eluted, and purified by using a QIAquick™ Gel extraction kit (QIAGEN, USA). The eluted DNA was precipitated with ethanol and resuspended in distilled water for ligation. As shown in
As shown in
A pET-28(+)a vector (NOVAGEN, Madison, Wis.) bearing a histidine-tag and a T7 promoter was digested with a restriction enzyme Ndel ( ENZYNOMICS, Korea). The pGEM-T Easy vector fragments containing the CP-Nm23 recombinant fragment and pET-28(+)a vector fragment were purified by using a QIAquick™ Gel extraction kit. Each of the pGEM-T Easy vector fragments was cloned into the pre-treated pET-28a(+) with a T4 ligase at 16° C. for 12 hours, followed by transformation of E. coli DH5α competent cells with the resulting pET-28a(+) vector (
After the clones were treated with the restriction enzyme NdeI (Enzynomics, Korea) and subjected to 0.8% agarose gel electrophoresis, it was verified that DNA fragments of about 0.5 kb for the full-length forms and vector fragments of about 5 kb were detected, confirming the cloning of the insert DNA of CP-Nm23 recombinant construct into pET-28a(+) vector, as shown in
The successfully cloned expression vectors for expressing cell permeable Nm23 recombinant proteins were designated pET28a(+)-HM1N, pET28a(+)-HNM1, pET28a(+)-HM1NM1, pET28a(+)-HM2N, pET28a(+)-HNM2, pET28a(+)-HM3N, pET28a(+)-HNM3 and pET28a(+)-HM3NM3, respectively. Among them, the E. coli transformants DH5α/HM3Nm23 and DH5α/HNm23M3 obtained by transforming E. coli DH5α with the expression vectors pET28a(+)-HM3N and pET28a(+)-HNM3, respectively, were deposited on Aug. 28, 2008 in accordance with the Budapest Treaty under accession numbers KCTC-11380BP and KCTC-11381BP with the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52, Oun-Dong, Yusong-Ku, Taejon 305-333, Republic of Korea.
The results of sequencing analysis are as follows:
As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a kFGF4-derived MTD, His-MTD1-Nm23 (HM1N) has an amino acid sequence represented by SEQ ID NO: 22, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 21; His-Nm23-MTD1 (HNM1) has an amino acid sequence represented by SEQ ID NO: 24, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 23; and His-MTD1-Nm23-MTD1 (HM1NM1) has an amino acid sequence represented by SEQ ID NO: 26, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 25.
As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a JO-76 MTD, His-MTD2-Nm23 (HM2N) has an amino acid sequence represented by SEQ ID NO: 28, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 27; and His-Nm23-MTD2 (HNM2) has an amino acid sequence represented by SEQ ID NO: 30, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 29.
As for the full-length forms of the cell permeable Nm23 recombinant protein constructed by using a JO-77 MTD as described above, His-MTD3-Nm23 (HM3N) has an amino acid sequence represented by SEQ ID NO: 32, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 31; His-Nm23-MTD3 (HNM3) has an amino acid sequence represented by SEQ ID NO: 34, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 33; and His-MTD3-Nm23-MTD3 (HM3NM3) has an amino acid sequence represented by SEQ ID NO: 36, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 35.
As a control for the cell permeable Nm23 recombinant proteins, His-Nm23 (HN), where a full-length Nm23 is fused only to a nuclear localization sequence (NLS) derived from SV40 large T antigen and a histidine-tag (His-Tag) without any MTD, was constructed. The control protein has an amino acid sequence represented by SEQ ID NO: 20, while a polynucleotide encoding the same has a nucleotide sequence represented by SEQ ID NO: 19.
<2-1>Selection of Optimal Bacterial Strains
To select the optimal bacterial strain for the expression of cell permeable Nm23 recombinant proteins prepared in Example 1 above, the following experiments were carried out in E. coli BL21(DE3), BL21-Gold(DE3), BL21-CodonPlus(DE3) and BL21-Gold(DE3) pLysS strains (STRATAGENE, USA), all of which contain the LacI promoter.
First, each of the expression vectors pET28a(+)-HM1N, pET28a(+)-HNM1, pET28a(+)-HM1NM1, and pHN (control) was transformed into E. coli BL21(DE3), BL21-Gold(DE3), BL21-CodonPlus(DE3) and BL21-Gold(DE3) pLysS strains, respectively, according to the heat shock method. Further, each of the expression vectors pET28a(+)-HM2N, pET28a(+)-HNM2, pET28a(+)-HM3N, pET28a(+)-HNM3 and pET28a(+)-HM3NM3 was transformed into E. coli BL21-Gold(DE3) strain, respectively, according to the heat shock method. After the transformation, the cells were cultured in an LB agar plate containing 50 μg/ml of kanamycin. Colonies formed on the plate were grown in 1 in of LB medium at 37° C. overnight, followed by culturing at 37° C. in 100 μl of LB medium with vigorous shaking until the optical density 600 (OD600) reached 0.5. IPTG (isopropyl-β-D-thiogalactoside) was then added thereto at a final concentration of 0.7 mM to induce the expression of the CP-Nm23 recombinant proteins. Protein induction was prolonged for 2 hours at 30° C. The E. coli culture solutions were harvested by centrifugation at 4° C., 7,000×g for 20 minutes, resuspended in a lysis buffer (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, pH 8.0), and subjected to ultrasonication on ice using a sonicator equipped with a probe. The cell lysates were centrifuged at 14,000×g for 15 minutes, so as to separate an insoluble fraction from a soluble fraction. The thus obtained soluble and insoluble fractions of CP-Nm23 recombinant proteins expressed in the E. coli strain with IPTG were loaded on a SDS-PAGE gel.
As shown in
<2-2>Expression of Recombinant Proteins
Each of the expression vectors pET28a(+)-HM1N, pET28a(+)-HNM1, pET28a(+)-HM1NM1, pET28a(+)-HM2N, pET28a(+)-HNM2, pET28a(+)-HM3N, pET28a(+)-HNM3 and pET28a(+)-HM3NM3 was transformed into E. coli BL21-Gold(DE3), selected as the optimal strain in section <2-1> of Example 2 above, followed by inducing their expression through the addition of 0.7 mM IPTG, according to the same method described in section <2-1> of Example 2. After that, soluble and insoluble fractions of CP-Nm23 recombinant proteins obtained therefrom were loaded on a SDS-PAGE gel.
As shown in
<3-1>Purification of Recombinant Proteins
The inducible expression of cell permeable Nm23 recombinant proteins in an E. coli system leads to the formation of insoluble aggregates, which are known as inclusion bodies. To completely solubilize these inclusion bodies, all of the above expressed proteins were denatured by dissolving them in 8 M urea used as a strong denaturing agent.
First, the BL21 Gold(DE3) strains transformed with each of the expression vectors pET28a(+)-HM1N, pET28a(+)-HNM1, pET28a(+)-HM1NM1, pET28a(+)-HM2N, pET28a(+)-HNM2, pET28a(+)-HM3N, pET28a(+)-HNM3, pET28a(+)-HM3NM3 and pET28a(+)-HM (control) were cultured in 1 l of an LB medium as described in Example 2. Each culture solution was harvested by centrifugation, gently resuspended in 20 of a lysis buffer (HN and HNM1: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0; other CP-Nm23: 100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, pH 8.0) without forming bubbles, and subjected to ultrasonication on ice using a sonicator equipped with a microtip. The cells were intermittently sonicated for 30 seconds, followed by cooling for 10 seconds, while setting the power to 25% of the maximum power. The total sonication time was 5 minutes. The cell lysates were centrifuged at 4° C., 4,000×g for 20 minutes, so as to separate the supernatant and the cellular debris pellet. The supernatant was loaded onto a Ni-NTA agarose resin where nitrilotriacetic acid agarose was charged with nickel (Ni). The Ni-NTA agarose resin was equilibrated with the lysis buffer. The supernatant was allowed to absorb onto the resin by gently shaking (using a rotary shaker) at 4° C. for 8 hours or more. The resin absorbed with the inclusion bodies containing the recombinant protein was centrifuged at 4° C., 1,000×g for 5 minutes, to remove the reaction solution and washed with a washing buffer (HN and HNM1: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 6.3; other CP-Nm23: 100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, pH 6.3) five times to remove nonspecific absorbed materials. After washing, the proteins absorbed to the resin were eluted with an elution buffer (HN and HNM1: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 4.5; other CP-Nm23: 100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, pH 4.5) with stirring for 2 hours or more under acidic conditions of pH 4.5. The eluted proteins were analyzed with 12% SDS-PAGE gel electrophoresis, stained with Coomassie Brilliant Blue R by gently shaking, and destained with a destaining solution.
According to the results shown in
<3-2>Refolding of Recombinant Proteins
Since the cell permeable Nm23 recombinant proteins of the present invention purified from the insoluble fraction as described in section <3-1> of Example 3 above were denatured by a strong denaturing agent, such as 8 M urea, the denatured proteins must be converted into an active form by a refolding process, as follows.
First, the purified recombinant proteins were subjected to a refolding process by dialyzing them against a refolding buffer (0.55 M guanidine HCl, 0.88 M L-arginine, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 100 mM NDSB, 1 mM glutathione oxidized, and 1 mM glutathione reduced) at 4° C. for 24 hours, thereby removing the denaturing agent. All of the refolded recombinant proteins were dialyzed against a cell culture medium DMEM (Dulbecco's Modified Eagle Medium) by using a dialysis bag (Snakeskin pleated, PIERCE) at 4° C. for 10 hours while stirring. The medium was replaced with fresh DMEM every 3 hours. The cell permeable Nm23 recombinant proteins of the present invention converted into their active form through the refolding process were used in the following experiments.
In order to quantitatively determine the cell permeability of the cell permeable Nm23 recombinant proteins according to the present invention, the introduction of the proteins into the cell was analyzed by FACS (fluorescence-activated cell sorting) in an animal model, as follows.
The cell permeable Nm23 recombinant proteins refolded into their active form in section <3-2> of Example 3 above were labeled with FITC (fluorescein-5-isothiocyanate, MOLECULAR PROBE). The recombinant protein (2 to 20 mg) was mixed with 1 μl of FITC at a concentration of 333 mg/ml and reacted in a dark room at room temperature for 1 hour with gentle stirring. The reaction solution was subjected to a dialysis against DMEM at 4° C. for 2 days until the unreacted FITC was completely removed, thereby obtaining FITC-conjugated recombinant proteins. Thus obtained FITC-conjugated recombinant proteins were subjected to a Bradford protein assay to measure the protein concentration. As a result, each of the FITC-conjugated recombinant proteins was measured to have a concentration of about 0.7 μg/μl.
Meanwhile, RAW 264.7 cells derived from mouse macrophage were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (500 mg/ml) and incubated at 37° C. in a humidified atmosphere of 5% CO2 in air.
After the incubation, the cells were treated with 10 μM of each of the FITC-conjugated recombinant proteins (HM1N, HNM1, HM1NM1, HM3N, HNM3 and HM3NM3) prepared above, followed by further culturing them for 1 hour at 37° C. Subsequently, the cells were treated with trypsin/EDTA (T/E, INVITROGEN) to remove cell surface bound proteins, washed with cold PBS (phosphate buffered saline) three times, and then, subjected to flow cytometry analysis by using a CELLQUEST PRO software program of the FACS (fluorescence-activated cell sorting) Calibur system ( BECKTON-DICKINSON). The cell concentration of each sample was 1×104 cells/μl, and the analysis was performed twice or more. The cell permeability of the cell permeable Nm23 recombinant proteins according to the present invention was determined by comparing it to that of the control protein (HN) not fused to a MTD.
a and 7b show the results of the flow cytometry analysis where the gray filled curve represents cell only, the black curve represents FITC only, the blue curve represents the cell permeability of the control protein not fused to a MTD (HN), the red curve represents the cell permeability of the cell permeable recombinant proteins HM1N, HM3N, HNM3 and HM3NM3, the green curve represents the cell permeability of the cell permeable recombinant protein HNM1, and the orange curve represents the cell permeability of the cell permeable recombinant protein HM1NM1. Referring to the results shown in
To visualize the intracellular localization of human Nm23 proteins delivered into a cell, NIH 3T3 cells (Korean Cell Line Bank, Seoul, Republic of Korea) were treated with FITC-conjugated recombinant proteins (HM1N, HNM1, HM1NM1, HM2N, HNM2, HM3N, HNM3 and HM3NM3) and visualized by confocal laser scanning microscopy.
First, the NIH 3T3 cells were cultured in an 8-well chamber slide (LabTek, Nalgen Nunc) for 24 hours. The NIH3T3 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 5% penicillin/streptomycin (500 mg/ml) in 5% CO2 at 37° C. After the cells were washed with PBS three times, the cells were treated with serum-free DMEM, serum-free DMEM containing FITC, and serum-free DMEM containing 10 μM of each of FITC-conjugated recombinant proteins, respectively, in 5% CO2 at 37° C. One hour later, the cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes.
For a direct detection of FITC-conjugated recombinant proteins that were internalized, the cells were washed with PBS three times and counterstained with a nuclear fluorescent stain solution, propidium iodide (PI, SIGMA-ALDRICH). The cells were stained with PI at a concentration of 1 μg/ml for 5 minutes, followed by washing with PBS three times. In order to preserve the FITC fluorescence of the recombinant protein, the glass slide was fixed in 10 μl of a polyvinyl alcohol mounting medium containing DABCO (Fluca) for 15 minutes before the observation. The intracellular distribution of the fluorescence was determined at the middle of a single cell analyzed by a confocal laser scanning microscope using a normaski filter. The confocal laser scanning microscopy was employed for observing cell phormology, FITC fluorescence and PI fluorescence. FITC was excited at 488 nm and detected by means of a bandpass filter at 530 nm.
As shown in
In order to confirm the in vivo function of the cell permeable Nm23 recombinant proteins according to the present invention, the biochemical functions of the recombinant proteins were examined on three types of cancer cell lines by Western blot analysis.
MDA-MB-435 and MDA-MB-231 cells, the highly metastatic human breast cancer cell lines used in this experiment, were purchased from Korean Cell Line Bank (Seoul, Republic of Korea). The cell lines were maintained in a RPMI 1640 medium (L-glutamine 300 mg/l, 25 mM HEPES and 25 mM NaHCO3) supplemented with 10% FBS and 1% penicillin/streptomycin in a 5% CO2 incubator at 37° C. CCL-185 cells, a human lung cancer cell line, were obtained from ATCC and maintained in a HamF-12K medium (2 mM L-glutamine, 1500 mg/f sodium bicarbonate) supplemented with 10% FBS and 1% penicillin/streptomycin in a 5% CO2 incubator at 37° C.
After 2 ml of the RPMI 1640 medium supplemented with FBS was added to each well of a 6-well plate, MDA-MB-435, MDA-MB-231, and CCL-185 cells were inoculated thereto at a concentration of 5×106 cells/ml. The well plate was incubated at 37° C. for 1 day so as to allow the cells to grow while adhering to the well plate. After removing the medium, the cells adhered to the well plate were washed with cold PBS. Subsequently, the cells were treated with 500 μl of each of the cell permeable Nm23 recombinant proteins and MTD-lacking Nm23 control protein (HIN) at a concentration of 10 μM, and reacted in a 5% CO2 incubator at 37° C. for 1 hour. The MDA-MB-435 cells were treated with each of HM1N, HNM1, HM1NM1, HM2N, HNM2, HM3N, HNM3 and HM3NM3 recombinant proteins, while MDA-MB-231 and CCL-185 cells were treated with each of HM3N, HNM3 and HM3NM3 recombinant proteins. After the reaction was completed, the cells were washed twice with PBS, and then, cultured in the presence of serum under the same conditions noted above for 2, 4, 6 and 8 hours, respectively.
After the cultivation was completed, the cells were resuspended in 200 μl of a lysis buffer (20 mM HEPES, pH 7.2, 1% Triton-X, 10% glycerol and proteinase inhibitor) and subjected to ultrasonication on ice for 30 minutes, to thereby obtain a cell lysate. The cell lysate was centrifuged at 12,000 rpm for 20 minutes at 4° C. to separate the supernatant. The thus obtained supernatant was subjected to a Bradford protein assay to quantitatively measure the protein concentration. The recombinant protein was resuspended in a SDS-PAGE loading buffer at a concentration of 25 μM to prepare a cell lysate sample. The thus prepared cell lysate sample was heated at 90° C. for 5 minutes, and then, stored at −80° C. until use.
For the Western blot analysis, p21 (21 kDa, CELL SIGNALING TECHNOLOGY), phospho-p53 (Ser15, 53 kDa, Cell Signaling), phospho-MEK (Ser217/221, 45 kDa, CELL SIGNALING TECHNOLOGY), and phospho-Erk (Thr202/Tyr204, 42/44 kDa, CELL SIGNALING TECHNOLOGY) were used as primary antibodies, and goat anti-mouse IgG-HRP ( SANTA CRUZ BIOTECHNOLOGY) and goat anti-rabbit IgG-HRP (SANTA CRUZ BIOTECHNOLOGY) were used as secondary antibodies. The cell lysate sample was applied to a 12% SDS-PAGE at 100 V for 2 hours and transferred onto a polyvinylidene fluoride (PDVF) membrane at 100 V for 90 minutes. In order to prevent the nonspecific interaction between the blotted proteins and unrelated antibodies, the PVDF membrane was blocked with 5% non-fat dry milk in TBS/T (10 Mm Tris-Cl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) at room temperature for 1 hour. After removing the blocking buffer, the PVDF membrane was washed with TBS/T, followed by incubation with each of the primary antibodies (diluted with the freshly prepared blocking buffer at a ratio of 1:10000) for 1 hour at 4° C. After removing the primary antibody solution, the membrane was washed with TBS/T five times each for 5 minutes, and incubated with the secondary antibody (diluted with the freshly prepared blocking buffer at a ratio of 1:5000) for 1 hour at room temperature. After washing with TBS/T five times, the membrane was stained using an enhanced chemiluminescence (ECL) detection system (GE HEALTHCARE AMERSHAM UK) to visualize the antigen/antibody interaction.
As shown in
<7-1>Invasion assay
In order to examine whether tumor metastasis is inhibited by blocking cancer cell migration in cancer cells treated with the cell permeable Nm23 recombinant proteins according to the present invention, an invasion assay was carried out as follows.
First, a human breast cancer cell line, MDA-MB-435 cells, were cultured overnight in a RPMI 1640 medium supplemented with 10% FBS in the absence of growth factors. The next day, the cells were treated with trypsin and harvested, followed by suspension in the same RPMI 1640 medium. The cells were treated with each of the MTD-lacking Nm23 control protein (HN), and cell permeable Nm23 recombinant proteins (HM2N, HNM2, HM3N, HNM3 and HM3NM3) according to the present invention at a concentration of 10 μM at 37° C. for 1 hour. Meanwhile, the top part of a trans-well polycarbonate membrane filter (BD Falcon) having a pore size of 3 μm was coated with MATRIGEL (40 μg per each well; BD Biosciences). To the lower part of the chamber, a DMEM medium supplemented with 10% FBS was added as an adhesive substrate. The cells treated with the above protein were suspended in a DMEM medium supplemented with 0.1% FBS to prepare a cell suspension. The thus prepared cell suspension was inoculated on the trans-well membrane filter (1×105 cells per each well), and cultured in a 5% CO2 incubator at 37° C. for 20 to 24 hours. The filters were washed with PBS, and the non-invasive cells remaining on the surface of the upper part were removed by using a cotton swab. The invasive cells that passed through the Matrigel and migrated to the lower part of the filter were fixed with 4% paraformaldehyde for 5 to 10 minutes, and stained with 0.5% (w/v) hemacolor for 10 to 20 minutes. The number of cells migrated to the base surface of the membrane filter (violet color) was counted by observing with an optical microscope.
According to the results shown in
<7-2>Wound migration assay
In order to examine whether the cell permeable Nm23 recombinant proteins according to the present invention can inhibit the migration of a breast cancer cell line, MDA-MB-435 cells, having high migration activity, a wound migration assay was carried out as follows.
MDA-MB-435 cells were cultured in a 60-mm culture dish until they formed a confluent monolayer covering the bottom thereof. After incubation, the cells were treated with each of the MTD-lacking Nm23 control protein (HN) and cell permeable Nm23 recombinant proteins (HM2N, HNM2, HM3N, HNM3 and HM3NM3) according to the present invention at a concentration of 10 μM at 37° C. for 1 hour. After the cells were washed with PBS, they were wounded with a sterile yellow tip, to thereby form a reference line that separated the confluent area from the bare area. To the cells was added a RPMI medium (3 mL) supplemented with 10% FBS, followed by culturing in a 5% CO2 incubator at 37° C. for 24 hours. The cells were washed with PBS, fixed with methanol for 1 minute, stained with Giemsa (Chameleon Chemical) for 5 minutes, and then, washed with distilled water. The migration was quantified by counting the number of cells that migrated from the wound edge into the bare area with an inverted light microscope at 40× magnification.
Referring to the results shown in
In order to examine the in vivo inhibitory effect of the cell permeable Nm23 recombinant proteins on tumor metastasis which has already been confirmed in vitro, an immunohistochemical analysis was carried out as follows.
First, MDA-MB-435 cells, a highly metastatic human breast cancer cell line, were suspended in 0.1 of PBS at a concentration of 1×106 cells/ml and were injected to the outer tail vein of 5-week old MHC (major histocompatibility complex)-deficient Balb/c nu/nu mice. Twenty mice were subdivided into 4 groups of 5 mice each. Each of the cell permeable Nm23 recombinant protein (HM3N, 300 μg) to which a JO-77 MTD was fused, a vehicle (PBS, 300 μg) and MTD-lacking Nm23 control protein (HN, 300 μg), and an EGFP recombinant protein (HM3E) where a JO-77 MTD was fused to the N-terminus of EGFP was administered to the mice. Here, the MTD-fused EGFP recombinant protein was employed as a control to examine whether the JO-77 MTD being fused to Nm23 had an effect on Nm23 expression. Five weeks after MDA-MB-435 cells were injected to the mice, the proteins were administered daily to the mice of each group via intravenous injection for 21 days. After three mice were selected from each group and sacrificed, lung tissue samples were extracted therefrom. The other two mice remaining in each group had undergone further observation for 14 days after the administration was terminated, and then, lung tissue samples were extracted therefrom. The lung tissue samples were fixed with a Bouin fixation solution overnight for detecting metastatic colonies, washed with distilled water, and then embedded in paraffin to prepare a paraffin block. Thus prepared paraffin block was sliced with a microtome to have a thickness of 4 μm, where the slices were mounted on a glass slide and treated with xylene for 5 minutes three times to remove paraffin. The glass slide was subjected to immunohistochemical staining with vimentin as a metastatic marker.
For the immunohistochemical staining, anti-vimentin antibody (ABCAM) was employed as a primary antibody, and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) was used as a secondary antibody. In order to prevent the nonspecific interaction between blotted proteins and irrelevant antibodies, the glass slide was blocked with 5% non-fat dry milk in TBS/T (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) at room temperature for 1 hour with stirring. After removing the blocking buffer, the glass slide was washed with TBS/T three times, followed by incubating with the anti-vimentin antibody as a primary antibody (diluted with PBS at a ratio of 1:200) for 1 hour at 4° C. After the removing the primary antibody solution, the glass slide was washed with TBS/T five times each for 5 minutes, and incubated with the goat anti-mouse IgG-HRP as a secondary antibody (diluted with PBS at a ratio of 1:200) for 1 hour at room temperature. After washing with TBS/T (0.025% Triton-X 100) twice, the glass slide was stained with a DAB substrate to detect vimentin.
a shows the results of optically observing the lung tissue extracted from the mouse after the cell permeable Nm23 recombinant protein according to the present invention was administered for 21 days, and the same was extracted from the mouse where the administration of the cell permeable Nm23 recombinant protein was terminated for 14 days. As shown in
b depicts the results of immunohistochemical staining showing the expression of a metastatic marker, vimentin, in the lung tissue extracted from the mouse after the cell permeable Nm23 recombinant protein according to the present invention was administered for 21 days (Day 21), and the lung tissue extracted from the mouse where the administration of the cell permeable Nm23 recombinant protein was terminated for 14 days (Day 35). As shown in
In order to examine the effect of inducing apoptosis in tumor tissues after the administration of the cell permeable Nm23 recombinant proteins, a TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay was performed by using the same mouse model as described in Example 8. The TUNEL assay was carried out by using an in situ cell death detection kit (TMR red, ROCHE).
In particular, each of the cell permeable Nm23 recombinant protein (HM3N), vehicle and HN as a control, and MTD-fused EGFE recombinant protein (HM3E) was daily administered to the mice subdivided into four groups via intravenous injection for 21 days according to the same method as described in Example 8. After three mice were selected from each group and sacrificed, lung tissue samples were extracted therefrom. The other two mice remaining in each group had undergone further observation for 14 days after the administration was terminated, and then, lung tissue samples were extracted therefrom. The lung tissue samples were embedded in paraffin to prepare a paraffin block. Thus prepared paraffin block was sectioned with a microtome to have a thickness of 5 μm and mounted on a glass slide. The glass slide was treated with xylene for 5 minutes three times, to thereby remove paraffin. It was then successively treated with 100% ethanol twice for 5 minutes, and 90%, 80% and 70% ethanol each for 3 minutes so as to dehydrate the lung tissue, followed by incubation in PBS for 5 minutes. The glass slide was treated with 0.1% Trition® X-100 dissolved in a 0.1% sodium citrate solution for 8 minutes, and washed with PBS twice for 2 minutes. After a drop of TUNEL reaction buffer (50 μl, ROCHE, USA) was added to the glass slide, the glass slide was incubated in a humidified incubator at 37° C. for 1 hour, washed with PBS three times, and then, observed with a fluorescence microscope.
Referring to the results shown in
In order to examine the change in protein expression pattern in the tumor tissue treated with the cell permeable Nm23 recombinant protein according to the present invention, a microarray assay was performed as follows.
In particular, each of the cell permeable Nm23 recombinant protein (HM3N), vehicle and HN (control) was administered to the mice subdivided into three groups via intravenous injection for 21 days, and then left alone for 14 days after the administration was terminated, according to the same method as described in Example 9 above. Fourteen days after the administration was terminated, lung tissue samples were extracted from the mouse of each group and freezed with liquid nitrogen. Total RNA was isolated from the lung tissue by using a TRIZOL reagent (INVITROGEN) according to the manufacturer's instruction, and treated with an RNase-free DNase (LIFE TECHNOLOGIES, Inc.), to thereby completely remove the remaining genomic DNA.
The thus isolated RNA was subjected to synthesis and hybridization of a target cRNA probe by using a Low RNA Input Linear Amplification kit (Agilent Technology) according to the manufacturer's instruction. In brief, 1 μg of total RNA was mixed with a T7 promoter specific primer and reacted at 65° C. for 10 minutes. A cDNA master mix was prepared by mixing a first strand buffer (5×), 0.1 M DTT, 10 mM dNTP mix, RNase-Out and MMLV-RT (reverse transcriptase), and added to the reaction mixture. The resulting mixture was reacted at 40° C. for 2 hours, followed by reacting at 65° C. for 15 minutes, to thereby terminate the reverse transcription and dsDNA synthesis. A transcription master mix was prepared by mixing a transcription buffer (4×), 0.1 M DTT, NTP mix, 50% PEG, RNase-Out, inorganic pyrophosphatase, T7-RNA polymerase and cyanine (3/5-CTP) according to the manufacturer's instruction. The thus prepared transcription master mix was added to the dsDNA reaction mixture and reacted at 40° C. for 2 hours so as to perform dsDNA transcription. The thus amplified and labeled cRNA was purified with a cRNA Cleanup Module (AGILENT TECHNOLOGY) according to the manufacturer's instruction. The labeled target cRNA was quantified by using a ND-1000 spectrophotometer (NanoDrop Technologies, Inc.). After the labeling efficiency was examined, cRNA was mixed with a blocking agent (10×) and a fragmentation buffer (25×), and reacted at 60° C. for 30 minutes so as to carry out the fragmentation of cRNA. The fragmented cRNA was resuspended in a hybridization buffer (2×) and directly dropped on a Whole Human Genome Oligo Microarray (44K). The microarray was subjected to hybridization in a hybridization oven (Agilent Technology) at 65° C. for 17 hours, followed by washing according to the manufacturer's instruction (AGILENT Technology).
The hybridization pattern was read by using a DNA microarray scanner ( AGILENT Technology) and quantified by using a Feature Extraction Software ( AGILENT Technology). Data normalization and selection of fold-changed genes were carried out by using a Gene Spring GX 7.3 soft wear (AGILENT Technology). The average of the normalized ratio was calculated by dividing a normalized signal channel strength by a normalized control channel strength. Functional annotation for a gene was conducted by using a Gene Spring GX 7.3 software (AGILENT Technology) according to the Gene Ontology™ Consortium.
The results of the microarray analysis are summarized in
As described in Table 3 above, in case of the apoptosis-relating genes, while the expressions of Caspase 14, cell death-inducing DFFA-like effector c (Cidec), cell death-inducing DNA fragmentation factor and alpha subunit-like effector A (Cidea) were up-regulated by about 3.5-, 4.0-, 2.5- and 2.5-fold, respectively, in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.
As described in Table 4 above, in case of the cell adhesion-relating genes, the expression of cadherin-like 26 was down-regulated by about 3.0-fold in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.
As described in Table 5 above, in case of the cell cycle regulation-relating genes, the expression of Avian erythroblastosis virus E-26 (v-ets) oncogene was down-regulated by about 4.0-fold in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.
As described in Tables 6a and 6b above, in case of the cell growth-relating genes, while the expression of member 17 of a tumor necrosis factor receptor superfamily was up-regulated by about 6.8-fold, the expressions of palate, lung and nasal epithelium carcinoma associated genes were down-regulated by about 26.0-fold in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.
As described in Table 7 above, in case of the cell proliferation-relating genes, the expression of signal transducer and activator of transcription 6 was up-regulated by about 5-fold in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.
As described in Tables 8a and 8b above, in case of immune response-relating genes, the expressions of immunoglobulin heavy chain (J558 family), immunoglobulin heavy chain complex and immunoglobulin joining chain were up-regulated by about 18-, 15- and 30-fold, respectively, in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.
As described in Table 9 above, in case of metastasis-relating genes, the expressions of fascin homolog 1 (actin bundling protein), prostaglandin-endoperoxide synthase 2 and vascular cell adhesion molecule 1 were up-regulated by about 2.5-, 2.5- and 2.0-fold, respectively, in the mouse group treated with the cell permeable Nm23 recombinant protein compared to that treated with the control protein.
Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.
The cell permeable Nm23 recombinant proteins of the present invention can induce the KSR phosphorylation and inactivation and inhibit the Ras-mediated MAPK cascade by efficiently introducing a metastasis suppressor Nm23 into a cell. Therefore, the cell permeable Nm23 recombinant proteins of the present invention can be effectively used as an anti-metastatic agent capable of preventing cancer metastasis by suppressing the proliferation, differentiation and migration of cancer cells.
This application is a National Stage of PCT/KR08/005221 filed Sep. 4, 2008 and claims the benefit of U.S. 60/969,714 filed Sep. 4, 2007.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR2008/005221 | 9/4/2008 | WO | 00 | 3/3/2010 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2009/031835 | 3/12/2009 | WO | A |
| Number | Date | Country |
|---|---|---|
| 2 336 223 | Aug 2001 | CA |
| 1033405 | Sep 2000 | EP |
| 1 127 576 | Aug 2001 | EP |
| WO 9105793 | May 1991 | WO |
| WO 9534295 | Dec 1995 | WO |
| WO 9811232 | Mar 1998 | WO |
| WO 0175067 | Oct 2001 | WO |
| WO 02083179 | Oct 2002 | WO |
| WO 2008093982 | Aug 2008 | WO |
| Entry |
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| Number | Date | Country | |
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| 20100323971 A1 | Dec 2010 | US |
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| 60969714 | Sep 2007 | US |
