The present invention pertains to have (i) cell-permeable GPX7 (CP-GPX7) proteins as protein-based biotherapeutics, which are well-enhanced in their ability to transport biologically active GPX7 proteins across the plasma membrane, to increase in its solubility and manufacturing yield, and to induce anti-cancer effect in gastrointestinal track (GIT) (ii) polynucleotides that encode the same, and (iii) anti-cancer compositions that comprise the same.
Gastrointestinal track (GIT) cancer including gastric and oesophageal cancer is the most common cancer in Asian countries (e.g., Korea, Japan) and a leading cause of cancer death worldwide, provoking considerable effort to understand the pathogenesis of the disease and to develop improved methods for diagnosis and treatment. Therapeutic options are limited for GIT-cancer not cured by surgical resection, and over-all 5-year survival rates are in the range of 30%.
Gastro-oesophageal reflux disease (GORD) is a condition where gastric acid, usually mixed with bile acids, refluxes into the lower oesophagus. GORD-associated chronic mucosal injury and inflammation is a major risk factor for the development of Barrett's oesophagus (BO), a premalignant condition that is closely associated with the development of oesophageal adenocarcinoma (OAC). Patients with BO can progress to low-grade dysplasia (LGD), high-grade dysplasia (HGD), and OAC at 30 to 60 times that of the general population.
Gastric acid and/or in combination with bile acids has been reported to induce a significant increase in intracellular reactive oxygen species (ROS) in oesophageal epithelial cells. The ROS levels are known to be higher in tissues with Barrett's oesophagus and OAC. In addition, higher levels of oxidative DNA damage, as well as single and double strand breaks in human Barrett's oesophagus and OAC are also major characteristics of the GIT-cancers. Normal cells have intact anti-oxidative properties that protect cells from ROS-induced DNA damage.
Among these systems, the glutathione peroxidase (GPX) family is a major anti-oxidative enzyme family that catalyzes the reduction of hydrogen peroxide, organic hydroperoxide, and lipid peroxides by reduced glutathione. It has been recently reported a frequent dysfunction of glutathione peroxidase 7 (GPX7) in OAC and its precancerous BO and dysplasia, suggesting that impairment of the anti-oxidative capacity may contribute to the development of OAC. GPX7 is the first example of a novel class of tumor suppressor genes, which are of particular importance in the proximal digestive tract to limit adenocarcinoma growth. A recent discovery showed that GPX7 deficiency in mice leads to systemic oxidative stress, increased tumor suppressor functions in OAC. Loss of expression and dysfunction of GPX7 are frequent in OAC and its precancerous lesions. GPX7 hypermethylation is observed in cancer and Barrett's oesophagus, but not in adjacent normal squamous epithelium. Methylation of tumor suppressor pathway is emerging as a major anti-cancer mechanism across the entire tract.
In principle, protein-based therapeutics offer to a way to control biochemical processes in living cells under non steady-state conditions and with fewer off target effects than conventional small molecule therapeutics. In practice, systemic protein delivery in animals has proven difficult due to poor tissue penetration and rapid clearance. Protein transduction exploits the ability of some cell-penetrating peptide (CPP) sequences to enhance the uptake of proteins and other macromolecules by mammalian cells. Previously developed hydrophobic CPPs, named membrane translocating sequence (MTS), membrane translocating motif (MTM) and macromolecule transduction domain (MTD), are able to deliver biologically active proteins into a variety of cells and tissues. Various cargo proteins fused to these CPPs have been used to test the functional and/or therapeutic efficacy of protein transduction.
However, the recombinant proteins fused to previously developed hydrophobic CPPs displayed extremely low solubility, poor yields and relatively low cell- and tissue-permeability. Therefore, these recombinant proteins were not suitable for further clinical development as therapeutic agents. To overcome these limitations, cell-permeable GPX7 recombinant proteins (CP-GPX7) fused to the combination of novel hydrophobic CPPs, namely advanced macromolecule transduction domains (aMTDs) to greatly improve the efficiency of membrane penetrating ability in vitro and in vivo with solubilization domains to increase in their solubility and manufacturing yield when expressed and purified from bacteria cells.
In this new art of invention, aMTD/SDs-fused GPX7 recombinant proteins (CP-GPX7), much improved physicochemical characteristics (solubility & yield) and functional activity (cell-/tissue-permeability) compared with the proteins fused to previously developed hydrophobic CPPs. In addition, the newly developed CP-GPX7 has now been demonstrated to have therapeutic application in treating GIT cancer, exploiting the ability of GPX7 to suppress NF-κB signaling. The present invention represents that macromolecule intracellular transduction technology (MITT) enabled by the new hydrophobic CPPs that are aMTD may provide novel protein therapy through GPX7-intracellular protein replacement against the GIT cancer. These findings suggest that intracellular restoration of GPX7 with CP-GPX7 creates a new paradigm for anti-cancer therapy, and the intracellular protein replacement therapy with the GPX7 recombinant protein fused to the combination of aMTD and SDs pair may be useful to treat the cancer.
The present invention relates to cell-permeable GPX7 (CP-GPX7) recombinant proteins capable of mediating the transduction of biologically active macromolecules into live cells.
CP-GPX7 fused to novel hydrophobic CPPs—namely advanced macromolecule transduction domains (aMTDs)—greatly improve the efficiency of membrane penetrating ability in vitro and in vivo of the recombinant proteins.
CP-GPX7 fused to solubilization domains (SDs) greatly increase in their solubility and manufacturing yield when they are expressed and purified in the bacteria system.
The present invention also, relates to its therapeutic application for delivery of a biologically active molecule to a cell, involving a cell-permeable GPX7 recombinant protein, where the aMTD is attached to a biologically active cargo molecule.
Other aspects of the present invention relate to an efficient use of aMTD sequences for drug delivery, protein therapy, intracellular protein therapy, protein replacement therapy and peptide therapy.
The present invention provides cell-permeable GPX7 as a biotherapeutics having improved solubility/yield and cell-/tissue-permeability and anti-cancer effects in gastrointestinal track (GIT). Therefore, this would allow their practically effective applications in drug delivery and protein therapy including intracellular protein therapy and protein replacement therapy.
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings.
CP-GPX7 suppressed phosphorylation of RB, enhanced the expression of the p21 in gastric cancer cells. Gastric cancer cells (AGS and MKN74) were treated with the indicated 10 μM proteins for 24 hours and cell extracts were immunoblotted with antibody against p21, phospho-retinoblastoma tumor suppressor (RB) and β-actin.
Female Balb/c nu/nu mice were implanted with NCI-N87 tumor block (1 mm3) into left side of back. After tumor reached a size of 50-80 mm3 (start), the mice were injected daily (I.V.) for 3 weeks with the diluent alone, HSAG7SB or HSAM165G7SB (CP-GPX7) and observed for 2 weeks following the termination of the treatment.
In this invention, it has been hypothesized that exogenously administered GPX7 proteins could compensate for the apparent inability of endogenously expressed members of this physiologic regulator to interrupt constitutively active cancer-initiating NF-κB signaling and excessive cell cycle, resulting in the inhibition of the tumorigenesis. To prove our hypothesis, the GPX7 recombinant proteins were fused to novel hydrophobic CPPs called aMTDs to have high cell-/tissue-permeability, additionally adopted solubilization domains to increase their solubility/yield in physiological condition, and then tested whether exogenous administration of GPX7 proteins can reconstitute their endogenous stores and restore their basic function as the inhibitor that attenuates NF-κB signaling. This art of invention has demonstrated “intracellular protein therapy” by designing and introducing cell-permeable form of GPX7 that has a great potential of anti-cancer therapeutic applicability in gastrointestinal track.
1-1. Analysis of Previously Developed Hydrophobic CPPs
To address the limitation of previously developed hydrophobic CPPs, novel sequences have been developed. To design new hydrophobic CPPs for intracellular delivery of cargo proteins such as GPX7, identification of optimal common sequence and/or homologous structural determinants, namely critical factors (CFs), had been crucial. To do it, the physicochemical characteristics of previously published hydrophobic CPPs were analyzed. To keep the similar mechanism on cellular uptake, all CPPs analyzed were hydrophobic region of signal peptide (HRSP)-derived CPPs (e.g. membrane translocating sequence: MTS and macromolecule transduction domain: MTD) as explained previously.
These 17 hydrophobic CPPs published from 1995 to 2014 have been analyzed for their 11 different characteristics—sequence, amino acid length, molecular weight, pI value, bending potential, rigidity/flexibility, structural feature, hydropathy, residue structure, amino acid composition, and secondary structure of the sequences. Two peptide/protein analysis programs were used (ExPasy: http://web.expasy.org/protparam/, SoSui: http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html) to determine various indexes, structural features of the peptide sequences and to design new sequence. Followings are important factors analyzed.
Average length, molecular weight and pI value of the peptides analyzed were 10.8±2.4, 1,011±189.6 and 5.6±0.1, respectively.
Bending potential (Bending or No-Bending) was determined based on the fact whether proline (P) exists and/or where the amino acid(s) providing bending potential to the peptide in recombinant protein is/are located. Proline differs from the other common amino acids in that its side chain is bonded to the backbone nitrogen atom as well as the alpha-carbon atom. The resulting cyclic structure markedly influences protein architecture which is often found in the bends of folded peptide/protein chain. Eleven out of 17 were determined as ‘Bending’ peptide which means that proline should be present in the middle of sequence for peptide bending and/or located at the end of the peptide for protein bending. As indicated above, peptide sequences could penetrate the plasma membrane in a “bent” configuration. Therefore, bending or no-bending potential is considered as one of the critical factors for the improvement of current hydrophobic CPPs.
Since one of the crucial structural features of any peptide is based on the fact whether the motif is rigid or flexible, which is an intact physicochemical characteristic of the peptide sequence, instability index (II) of the sequence was determined. The index value representing rigidity/flexibility of the peptide was extremely varied (8.9-79.1), but average value was 40.1±21.9 which suggested that the peptide should be somehow flexible, but not too rigid or flexible.
Alanine (V), valine (V), leucine (L) and isoleucine (I) contain aliphatic side chain and are hydrophobic—that is, they have an aversion to water and like to cluster. These amino acids having hydrophobicity and aliphatic residue enable them to pack together to form compact structure with few holes. Analyzed peptide sequence showed that all composing amino acids were hydrophobic (A, V, L and I) except glycine (G) in only one out of 17 and aliphatic (A, V, L, I, and P). Their hydropathic index (Grand Average of Hydropathy: GRAVY) and aliphatic index (AI) were 2.5±0.4 and 217.9±43.6, respectively.
In the 11 characteristics analyzed, the following 6 are selected namely “Critical Factors (CFs)” for the development of new hydrophobic CPPs—advanced MTDs: i) amino acid length, ii) bending potential (proline presence and location), iii) rigidity/flexibility (instability index: II), iv) structural feature (aliphatic index: AI), v) hydropathy (GRAVY) and vi) amino acid composition/residue structure (hydrophobic and aliphatic A/a).
1-2. Analysis of Selected Hydrophobic CPPs to Optimize ‘Critical Factors’
Since the analyzed data of the 17 different hydrophobic CPPs (analysis A) previously developed during the past 2 decades showed high variation and were hard to make common- or consensus-features, additional analysis B and C was also conducted to optimize the critical factors for better design of improved CPPs-aMTDs.
In analysis B, 8 CPPs were used with each cargo in vivo. Length was 11±3.2, but 3 out of 8 CPPs possessed little bending potential. Rigidity/Flexibility was 41±15, but removing one [MTD85: rigid, with minimal (II: 9.1)] of the peptides increased the overall instability index to 45.6±9.3. This suggested that higher flexibility (40 or higher II) is potentially be better. All other characteristics of the 8 CPPs were similar to the analysis A, including structural feature and hydropathy.
To optimize the ‘Common Range and/or Consensus Feature of Critical Factor’ for the practical design of aMTDs and the random peptides (rPs or rPeptides), which were to prove that the ‘Critical Factors’ determined in the analysis A, B and C were correct to improve the current problems of hydrophobic CPPs—protein aggregation, low solubility/yield, and poor cell/tissue-permeability of the recombinant proteins fused to the MTS/MTM or MTD, and non-common sequence and non-homologous structure of the peptides, empirically selected peptides were analyzed for their structural features and physicochemical factor indexes.
The peptides which did not have a bending potential, rigid or too flexible sequences (too low or too high Instability Index), or too low or too high hydrophobic CPP were unselected, but secondary structure was not considered because helix structure of sequence was not required. 8 selected CPP sequences that could provide a bending potential and higher flexibility were finally analyzed. Common amino acid length is 12 (11.6±3.0). Proline should be presence in the middle of and/or the end of sequence. Rigidity/Flexibility (II) is 45.5-57.3 (Avg: 50.1±3.6). AI and GRAVY representing structural feature and hydrophobicity of the peptide are 204.7±37.5 and 2.4±0.3, respectively. All peptides are consisted with hydrophobic and aliphatic amino acids (A, V, L, I, and P). Therefore, analysis C was chosen as a standard for the new design of new hydrophobic CPPs (TABLE 1).
1-3. Determination of Critical Factors for Development of aMTDs
For confirming the validity of 6 critical factors providing the optimized cell-/tissue-permeability. all 240 aMTD sequences have been designed and developed based on six critical factors (TABLES 2-1 to 2-6). All 240 aMTDs (hydrophobic, flexible, bending, aliphatic and helical 12 a/a-length peptides are practically confirmed by their quantitative and visual cell-permeability. To determine the cell-permeability of aMTDs and rPeptides which do not satisfy one or more critical factors have also been designed and tested. Relative cell-permeability of 240 aMTDs to the negative control (random peptide (rP) 38, hydrophilic & non-aliphatic 12A/a length peptide) was significantly increased by up to 164 fold, with average increase of 19.6±1.6. Moreover, compared to reference CPPs (MTS/MTM1 and MTD), novel 240 aMTDs averaged of 13±1.1 (maximum 109.9) and 6.6±0.5 (maximum 55.5) fold higher cell-permeability, respectively. As a result, there were vivid association of cell-permeability of the peptides and critical factors. According to the result from the newly designed and tested novel 240 aMTDs, the empirically optimized critical factor (CFs) are provided below (TABLE 3).
These examined critical factors are within the range that we have set for our critical factors; therefore, we were able to confirm that the aMTDs that satisfy these critical factors have much higher cell-permeability and intracellular delivery potential compared to reference hydrophobic CPPs reported during the past two decades.
2. Development of GPX7 Recombinant Proteins Fused to aMTD and Solubilization Domain
2-1. Design of Novel Hydrophobic CPPs-aMTDs for Development of GPX7 Recombinant Proteins
Based on these six critical factors proven by experimental data, newly designed advanced macromolecule transduction domains (aMTDs) have been developed, and optimized for their practical therapeutic usage to facilitate protein translocation across the membrane. For this present invention, cell-permeable GPX7 recombinant proteins have been developed by adopting aMTD61 and aMTD165 (TABLE 5) that satisfied all 6 critical factors.
To determine the protein transduction activity of these aMTDs, histidine-tagged solubilization domain A (SDA)—a non-functional and soluble protein present in a spore surface coat of Myxococcus xanthus having tandem repeats of 2 N-terminal domain (NTD) sequences of CP—000113.1-recombinant proteins including aMTDs were designed. SDA recombinant proteins were conjugated to 5/6-fluorescein isothiocyanate (FITC) and cellular uptake of FITC-labeled SDA recombinant proteins were measured by flow cytometry (
2-2. Selection of Solubilization Domain for Recombinant Proteins
Since recombinant cargo proteins fused to previously developed hydrophobic CPP—MTS, MTM and MTDs—were highly insoluble and had extremely low solubility, we additionally adopted non-functional solubilization domains (SDs) to improve solubility, yield and stability of the recombinant proteins and hypothesized that fusion of GPX7 with SDs and novel hydrophobic CPP, aMTD, would greatly increase solubility/yield and cell-/tissue-permeability of recombinant cargo proteins for the clinical application. According to this specific aim, 5 solubilization domains were selected and information of these SDs are shown TABLE 6.
2-3. Preparation of Recombinant aMTD-GPX7-SD Fusion Proteins
We first designed 4 different kinds of recombinant proteins either with or without the aMTD61 and SDs for GPX7 recombinant proteins (set 1). Each recombinant protein of set 1 was named His-GPX7 (HG7), His-aMTD61-GPX7 (HM61G7), His-aMTD61-GPX7-SDA (HM61G7SA) and His-aMTD61-GPX7-SDB (HM61G7SB;
To develop stable CP-GPX7, aMTD61 and solubilization domains were replaced to aMTD165 and SDC fused to N terminus, respectively. Also, we designed 2 negative controls—recombinant proteins lacking GPX7 cargo protein (SDC-His-aMTD165; SCHM165) and aMTD (SDC-His-GPX7; SCHG7). In addition, solubilization domain was changed to SDD and SDF (
We subsequently generated GPX7 recombinant protein which containing both of SDA and SDB (HSAM165G7SB). In addition, GPX7 protein lacking aMTD (HSAG7SB) and GPX7 recombinant protein lacking GPX7 (HSAM165SB) were designed (
Yields per L of E. coli for each GPX7 recombinant protein (mg/L) ranged from 0.1 to 39 mg/L (
3. aMTD/SDs-Fused GPX7 Recombinant Proteins Significantly Increase Cell- and Tissue-Permeability
3-1. aMTD/SDs-Fused GPX7 Recombinant Proteins are Cell-Permeable
To examine the protein uptake, the GPX7 recombinant proteins were conjugated to FITC and cells were treated with 10 μM FITC-labeled GPX7 recombinant proteins. Internalized proteins were measured by flow cytometry (
3-2. aMTD/SDs-Fused GPX7 Recombinant Proteins Enhance the Systemic Delivery to a Variety of Tissues
To further investigate in vivo delivery of GPX7 recombinant proteins, FITC-labeled GPX7 proteins were monitored following intraperitoneal (IP) injections in mice. Tissue distributions of fluorescence-labeled-GPX7 proteins in different organs was analyzed by fluorescence microscopy (
3-3. Cell Permeability Mediated aMTD165 was Achieved Efficiently Depend on Stable Plasma Bi-Lipid Layer Membrane
We next investigated how of aMTD165-mediated intracellular delivery was occurred. The aMTD-mediated intracellular delivery of GPX7 protein did not require protease-sensitive protein domains displayed on the cell surface (
4. aMTD/SD-Fused GPX7 Protein Efficiently Inhibits Cellular Processes
4-1. CP-GPX7 Inhibited TNF-α Induced NF-κB Nuclear Translocation
The ultimate test of cell-penetrating efficiency is a determination of intracellular activity of GPX7 proteins transported by aMTD. Since GPX7 are known to inhibit the TNF-α-induced NF-κB activation in GIT-specific cancer, we demonstrated whether cell-permeable GPX7 inhibits the translocation of NF-κB into the nucleus. As shown in
4-2. CP-GPX7 Suppressed the Phosphorylation of NF-κB in Gastric Cancer Cells
We next investigated the effect of cell-permeable GPX7 proteins on phosphorylation of NF-κB. Treatment of human gastric cancer cells (AGS and MKN74) with GPX7 proteins containing aMTD165 and SDs (HSAM165G7SB) significantly suppressed the on phosphorylation of NF-κB (
5-1. CP-GPX7 Enhances the Penetration into Gastric Cancer Cells and Systemic Delivery to Stomach
To determine the cell-permeability of CP-GPX7 in the gastric cancer cells, cellular uptake of FITC-labeled GPX7 recombinant proteins was quantitatively evaluated by flow cytometry. FITC-HSAM165G7SB recombinant protein (CP-GPX7) promoted the transduction into cultured AGS and MKN75 gastric cancer cells (
5-2. CP-GPX7 Inhibits Cell Viability in GIT-Cancer Cells
Since the endogenous level of GPX7 protein is reduced in GIT-cancer cells and reconstitution of GPX7 significantly suppresses growth and proliferation of the cells, we therefore examined the effects of CP-GPX7 on survival in human gastric and oesophageal cancer cell lines. (
5-3. CP-GPX7 Regulates the Expression of Cell Cycle-Associated Proteins in Gastric Cancer Cells.
The CP-GPX7 appeared to be biological active, as HSAM165G7SB-treated cells expressed lower levels of phosphorylated retinoblastoma tumor suppressor (RB) and higher levels of p21, as compared to HSAG7SB-treated cells (
5-4. CP-GPX7 Induces Apoptosis in GIT-Cancer Cells
To further determine the effect of CP-GPX7 on the tumorigenicity of GIT-cancer cells, we subsequently investigated whether CP-GPX7 regulates apoptosis in gastric and oesophageal cancer cells. HSAM165G7SB protein (CP-GPX7) was a considerably efficient inducer of apoptosis in GIT-cancer cells, as assessed either by a fluorescent terminal dUTP nick-end labeling (TUNEL) assay (
5-5. CP-GPX7 Inhibits Migration of GIT-Cancer Cells
We next determined the ability of CP-GPX7 to influence migration. Human gastric cancer (AGS and MKN75) and oesophageal cancer (SK-GT-4) cells and mouse fibroblast (NIH3T3) were treated with 10 μM of GPX7 recombinant proteins for 2 hours, the monolayers were wounded, and cell migration in the wound was monitored after 24-72 hours (
We assessed the anti-tumor activity of CP-GPX7 against human gastric cancer xenografts. Balb/c nu/nu mice were subcutaneously implanted with tumor block (1 mm3) of gastric cancer cells into the left side of the back. Tumor-bearing mice were intravenously administered HSAM165G7SB or control proteins (HSAG7SB; 600 μg/head, respectively) for 21 days and observed for 2 weeks following the termination of the treatment (
The following examples are presented to aid practitioners of the invention, to provide experimental support for the invention, and to provide model protocols. In no way are these examples to be understood to limit the invention.
His-tagged GPX7 recombinant proteins were designed into the expression vectors which contain human GPX7 proteins fused with aMTD61 or aMTD165 and solubilization domain A (SDA) and solubilization domain B (SDB). The GPX7 sequence was amplified using human genomic DNA as a template. GPX7 and solubilization domains were constructed using primer sets (TABLES 7, 8 and 9) by polymerase chain reaction (PCR).
PCR using 100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1× reaction buffer and 0.5 U Pfu(+) DNA polymerase (MGmed, Seoul, Korea) is following three steps: (i) denaturation (95° C.) for 30 seconds, (ii) annealing (60° C.) for 30 seconds (iii) extension (72° C.) for 1 min each and these steps are repeated 35 times. Set 1 PCR products was cloned at NdeI (5′) and SalI (3′) in pET-28a (+) vector (Novagen, Darmstadt, Germany). Set 2 PCR products was cloned at BamHI (5′) and XhoI (3′) in pET-32a (+) vector (Novagen, Darmstadt, Germany). Set 3 PCR products was cloned at BamHI (5′) and XhoI (3′) in pET-39b (+) vector (Novagen, Darmstadt, Germany) and EcoRI (5′) and XhoI (3′) in pH6HTC His6HaloTag®T7 vector (Promega, Madison, Wis., USA). DNA segments and vectors were cleaved with restriction enzyme (NEB, Hertfordshire, UK) at 37° C. for 3 hours. Digested vectors were ligated with amplified and digested DNA fragments using T4 DNA ligase (NEB, Hertfordshire, UK) at 4° C. overnight. These ligated samples were added 5 μl to competent E. coli DH5-alpha cells (MGmed, Seoul, Korea) on ice for 10 minutes. It moved into the 42° C. water bath for 90 seconds and returned the tubes to ice and incubated for 10 minutes. Then, the mixture added with LB broth media was incubated in 37° C. shaking incubator for 1 hour. The entire culture was plated on LB broth agar plate with kanamycin (25 μg/mL) (Biopure reagents, Seoul, Korea) before incubating overnight at 37° C. Single colony was picked and make a plasmid prep. After the digestion of restriction enzymes, digested plasmid was confirmed by using 1% agarose gels electrophoresis (
The histidine-tagged GPX7 recombinant proteins were expressed from E. coli BL21-Gold (DE3) competent cells (Agilent Technologies, Santa Clara, USA) grown to an A600 of 0.7 and induced 16 hours at 25° C. with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant proteins (HSAM165G7SB and HSAM165SB) were lysed with lysis buffer A (10 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0), and purified by Ni2+ affinity chromatography. Column bound to proteins were washed three times with 30 ml of washing buffer A (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). Ni+2 affinity purified proteins were eluted three times with 20 mL of elution buffer A (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0).
Other recombinant proteins (HG7, HM61G7, HM61G7SA, HM61G7SB, SCHG7M165 and HSAG7SB) were expressed from E. coli BL21-Gold (DE3) competent cells grown to an A600 of 0.7 and induced 3 hours at 37° C. with 0.7 mM IPTG. Recombinant proteins were lysed using lysis buffer B (8 M Urea, 10 mM Tris, 100 mM NaH2PO4) and purified by Ni2+ affinity chromatography. Resin bound to proteins were washed 3 times with 30 ml of washing buffer B (8 M Urea, 10 mM Tris, 20 m imidazole, 100 mM NaH2PO4). Proteins were eluted 3 times with 30 mL of elution buffer B (8 M Urea, 10 mM Tris, 250 mM imidazole). After purification, they was dialyzed twice against a refolding buffer (550 mM Guanidine-HCl, 440 mM L-Arginine, 50 mM Tris, 100 mM Non-Detergents Sulfobetaines (NDSB), 150 mM NaCl, 2 mM reduced glutathione and 0.2 mM oxidized glutathione). Finally, they were dialyzed against a physiological buffer such as DMEM at 4° C. until the dialysis was over 300×105 times.
After purification, all proteins were dialyzed against DMEM as indicated above. Finally, SDS-PAGE analysis was conducted to confirm the presence of target protein and concentration of purified proteins was quantified using Bradford assay according to the manufacturer's instructions.
Solubility is scored on a 5 point scale ranging from highly soluble proteins with little tendency to precipitate (+++++) to largely insoluble proteins (+) by measuring their turbidity (A450). Yield (mg/L) in physiological buffer condition of each recombinant protein also is determined.
For quantitative cell permeability, the GPX7 recombinant proteins were conjugated to FITC according to the manufacturer's instructions (Pierce Chemical, Rockford, Ill.). RAW 264.7 cells and human gastric cancer cells (AGS and MKN75) were treated with 10 μM FITC-labeled proteins for 1 hour at 37° C., washed with cold PBS three times, treat with proteinase K (10 μg/ml) for 5 minutes at 37° C. to remove cell-surface bound proteins. Cell-permeability of recombinant GPX7 proteins was analyzed by flow cytometry (Guava, Millipore, Darmstadt, Germany).
For visual cell permeability, NIH3T3 cells were treated with 10 μM FITC-conjugated recombinant proteins for 1 hour at 37° C., washed with cold PBS three times, and treated with proteinase K (10 μg/ml) for 5 minutes at 37° C. Treated cells were fixed in 2% paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 30 minutes at room temperature, washed with cold PBS three times, and mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, Calif.) with DAPI (4′,6-diamidino 2-phenylindole) for nuclear staining. The intracellular localization of the fluorescent signal is determined by confocal laser scanning microscopy (LM700, Zeiss, Germany).
750 μg of FITC-labeled GPX7 recombinant proteins is administered to wild type Balb/c mice. Two hours later, the mice are sacrificed, and liver, kidney, spleen, lung, heart, brain and stomach were isolated. Isolated tissues were washed and frozen with an OCT compound (Sakura, Alphen an den Rijn, Netherlands) on dry ice, and then sectioned to a thickness of 20 μm. The tissue specimens are mounted on a glass slide and observed by fluorescence microscopy (Nikon, Tokyo, Japan).
The GPX7 recombinant proteins were conjugated to FITC according to the manufacturer's instructions (Pierce Chemical, Rockford, Ill.). Human gastric cancer cells (AGS) were pretreated with different agents to assess the effects of various conditions on the protein uptake: (i) 5 μg/ml proteinase K for 5 minutes, (ii) 20 μM Taxol for 30 minutes, (iii) 10 μM antimycin in the presence or absence of 1 mM ATP for 2 hours, (iv) incubation on ice (or maintained at 37° C.) for 15, 30 or 60 minutes, and (v) 100 mM EDTA for 3 hours. Cells were then incubated with 10 μM FITC-labeled GPX7 recombinant proteins for 1 hour at 37° C., washed three times with cold PBS and treated with proteinase K (10 μg/ml) for 10 minutes at 37° C. to remove the cell-surface bound proteins and cell-permeability of GPX7 recombinant proteins was analyzed by flow cytometry (Guava, Millipore, Darmstadt, Germany).
Cell viability was evaluated with the CellTiter-Glo luminescent cell viability assay (Promega, Madison, Wis., USA). Human gastric cancer cells (AGS, MKN75, MKN74 and MKN45) and human oesophageal cancer cells (FLO-1, OE19 and OE33) were treated with 10 μM GPX7 recombinant proteins (HSAM165G7SB, HSAG7SB and/or HSAM165SB) or buffer alone with 2% fetal bovine serum for 72 hours at 37° C. Then, add a volume of CellTiter-Glo reagent equal to the volume of cell culture medium present in each well and incubate for 10 minutes at room temperature, record luminescence using microplate luminometer (LUMIstar Omega, BMG LABTECH, Ortenberg, Germany). The results were relative cell viability compared with the vehicle (buffer alone with 2% FBS)-treated controls.
Cancer cell migration was determined using the wound healing assay. Briefly, cells were seeded into 12-well plates and grown to 90% confluence. The wounds were produced by scraping of the cell layer with a sterile white tip. Cells were treated with 10 μM GPX7 recombinant proteins (HSAM165G7SB, HSAG7SB and/or HSAM165SB) or buffer alone for 2 hours prior to changing the growth medium. Cells were cultured for an additional 24 hours (AGS, SK-GT-4 and NIH3T3) or 72 hours (MKN75) at 37° C. before being photographed. The migration is quantified by counting the number of cells that migrated from the wound edge into the clear area.
Determination of Catalytic Effects of aMTD/SD-Fused GPX7 Recombinant Proteins on GIT-Cancer Cell Apoptosis
Apoptotic cells were analyzed using TUN EL assay with In Situ Cell Death Detection kit TMR red (Roche, 4056 Basel, Switzerland). Human gastric cancer cells (AGS and MKN45) and human oesophageal cancer cells (OE33) were treated with 10 μM GPX7 recombinant proteins (HSAM165G7SB and HSAG7SB) or buffer alone for 24 hours with 2% fetal bovine serum, washed with cold PBS two times. Treated cells were fixed in 4% paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 1 hour at room temperature, washed with cold PBS twice and incubated in the permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate solution) for 2 minutes on the ice. Cells were washed with cold PBS twice, and treated with TUNEL reaction mixture for 1 hour at 37° C. in dark, washed with cold PBS three times and observed by fluorescence microscopy (Nikon, Tokyo, Japan).
Human gastric cancer cells (AGS and MKN45) were grown on glass cover slips to approximately 80% confluency, treated with 10 μM GPX7 recombinant proteins (HSAM165G7SB, HSAG7SB and HSAM165SB) or buffer alone for 2 hours at 37° C., and washed cold PBS twice. Cells on the coverslips were treated with human TNF-α (50 ng/ml) for 30 minutes, fixed in 4% paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 15 minutes at room temperature, washed cold PBS three times. The coverslips were incubated with 0.1% Triton X-100 (Biopure reagents, Seoul, Korea) for 30 minutes at room temperature, incubated with primary antibody against NF-κB-p65 (1:100, Cell Signaling Technology, Boston, USA) in 5% bovine serum albumin in TBS-T for overnight at 4° C. Secondary antibody was Texas Red®-X goat anti rabbit IgG (1:500, Life technologies, California, USA). The coverslips were mounted on to glass slides using VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, Calif.) with DAPI (4′,6-diamidino 2-phenylindole) for nuclear staining and observed by fluorescence microscopy (Nikon, Tokyo, Japan).
Human gastric cancer cells (AGS and MKN74) were cultured in RPMI-1640 medium and maintained at 37° C. in an atmosphere containing 5% CO2. A total of 5×106 cells were treated with 10 μM GPX7 recombinant proteins (HSAM165G7SB and HSAG7SB) or buffer alone for 24 hours. Cells were lysed in a RIPA buffer containing EDTA-free protease inhibitor cocktail (Roche, 4056 Basel, Switzerland) and Halt™ phosphatase inhibitor cocktail (Thermo, Rockford, USA). Equal amounts of cell lysate protein were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The protein transferred membranes were incubated to block non-specific binding sites in immersing the membrane in 5% non-fat dried milk. The membranes were incubated with the rabbit anti-phospho RB antibody, the rabbit anti-phospho NF-κB p65 antibody, the rabbit anti-p21 antibody and the rabbit anti-β-actin antibody diluted 1:1000 in 5% bovine serum albumin in TBS-T at 4° C. for 24 hours. Secondary antibody was anti-rabbit IgG-HRP diluted 1:5000 in 5% skim milk in TBS-T for 1 hour at room temperature. All antibodies were purchased from Cell Signaling Technology (Boston, USA). The blots were developed using a chemiluminescence detection system, SuperSignal® West Dura Extended Duration Substrate (Thermo, Rockford, USA) and exposed to an x-ray film.
It will be apparent to those skilled in the art that various modifications can be made to the above-described exemplary embodiments of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention covers all such modifications provided that they come within the scope of the appended claims and their equivalents.
This application claims the benefit of the filing date of U.S. Provisional Application No. 62/046,067, filed on Sep. 4, 2014, in the United States Patent and Trademark Office, the disclosure of which is incorporated herein in its entirety by reference.
Number | Date | Country | |
---|---|---|---|
62046067 | Sep 2014 | US |