KR20060088569A - Recombinant protein, its coding gene, and use for cancer suppression activity - Google Patents

Abstract

The present invention discloses a recombinant protein having an anticancer effect, a coding gene thereof, and a method of using the same. Recombinant protein having an anticancer effect according to the present invention is 1) a protein having an amino acid sequence of SEQ ID 2 in the sequence listing, 2) a protein derived from SEQ ID 2 and having at least 90% homology with SEQ ID 2 3) a protein derived from SEQ ID 2, which is obtained by substituting, deleting or adding one or several amino acid residues in the amino acid sequence of SEQ ID 2 and having the same activity as SEQ ID 2; One. Drugs having the above-mentioned recombinant protein as an active ingredient have a large selective inhibitory effect on tumor cells and have an important application value without inducing apoptosis of normal tissue cells.

Description

Recombinant Protein with Cancer Suppression Action, Its Encoding Gene and Use}

The present invention relates to the fields of genetic engineering and pharmacy. The present invention relates to a drug of genetic engineering that contains a recombinant protein, a coding gene, and a recombinant protein as an active ingredient. In particular, the present invention relates to a therapeutic protein for cancer comprising a recombinant protein, an encoding gene, and the recombinant protein having an anticancer effect as an active ingredient.

In evolution, mammals gradually establish a pair of signaling mechanisms for apoptosis that can induce the planned death of individual cells. The underlying theory is that ligands in lethal cells interact with death receptors on the cell surface to induce cell apoptosis. This beneficial apoptosis plays an important physiological role in the removal of activated lymphocytes at the end of the immune response and in the removal of infected cells and cells modified to have oncogenes. Examples of this include the interaction between TNF and receptor TNFR, and the interaction between FasL and receptor Fas / Apo1 / CD95.

Activated death receptors are directly involved in cascade reactions of cell apoptosis, which can induce apoptosis of various cancer cells and are potential anticancer factors. TNF and FasL can induce apoptosis of cancer cells, but they can cause serious toxicity and side effects in chemotherapy. Inoculation of TNF can result in a reaction that causes fatal inflammation similar to shock from infection. This response is mainly mediated by NF-kB, a transcription factor present in vascular endothelial cells and macrophages and activated by TNF. Fas inhibitory antibodies can induce apoptosis of Fas dependent cells present in liver cells resulting in fatal liver damage.

Wiley et al. (1995) discovered a ligand (TRAIL) that induces TNF-related apoptosis based on sequence matching of TNF and FasL in 1995. TRAIL can induce apoptosis by interacting with death receptors DR4 or DR5. Unlike TNF and FasL, mRNA of TRAIL is continuously expressed in many normal human tissues. While they share normal cells, they represent a physiological mechanism that induces apoptosis of cancer cells. These mechanisms include expression of inhibitory receptors, competitive with DR4 and DR5 to bind ligands, and the ability of TRAIL to interact with three receptors DcR1, DcR2 and DPG. Therefore, the toxicity of TRAIL is less than that of TNF and FasL.

1 shows curves of tumor volume of human glioma U251 under various treatment conditions.

2 shows tumor size of human glioma U251 under various treatment conditions.

3 shows a curve of tumor volume of human lung cancer NCI-H460 under various treatment conditions.

4A to 4F show the histopathological portion of human lung cancer NCI-H460 under various treatment conditions. (HE dyeing * 100)

5 shows tumor size of human lung cancer NCI-H460 under various treatment conditions.

6 shows a curve of tumor volume of human colon cancer COLO 205 under various treatment conditions.

7A to 7E show BALB / c-nu nude mice inoculated with human colon cancer COLO 205 followed by various treatments.

It is an object of the present invention to provide a drug for effectively treating a cancer comprising a recombinant protein having an anticancer effect and a recombinant protein as an active ingredient.

Recombinant protein having an anticancer effect according to the present invention can be selected from the following.

1) a protein having the amino acid sequence of SEQ ID 2 in the sequence list

2) A protein derived from SEQ ID No. 2 and a protein having at least 90% homology with SEQ ID No. 2 and having the same activity.

3) A protein derived from SEQ ID 2, obtained by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of SEQ ID 2 and having the same activity as SEQ ID 2

The protein of SEQ ID 2 in the sequence listing is a human recombination of cyclically permuted TRAIL (CPT), consisting of 166 amino acid residues.

The active ingredient of the drug for treating cancer (CPT) provided by the present invention is one of the aforementioned proteins.

If desired, one or more pharmaceutically acceptable carriers may be added to the drug. Carriers include diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption enhancers, surfactants, absorbent carriers, brighteners commonly used in the pharmaceutical art. If necessary, flavors, sweeteners and the like may be added.

The medicine according to the invention can be made in various forms, namely inoculated solutions, tablets, powders, powders, capsules, liquids taken by mouth, ointments or creams and the like. All dosage forms mentioned above may be prepared by conventional methods in the pharmaceutical art.

The coding sequence of the active element (protein) of the drug for treating cancer (CPT) provided by the present invention can be selected from the following.

1) SEQ ID 1 of the sequence list

2) a polynucleotide capable of encoding the amino acid sequence of SEQ ID 2 in the sequence listing

3) A DNA sequence having more than 90% sequence homology with the DNA sequence defined by SEQ ID 1 in the Sequence Listing, which encodes a protein having the same action as the protein encoded by SEQ ID 1.

4) A DNA sequence encoding a protein derived from SEQ ID 2, wherein the protein derived from SEQ ID 2 is obtained by substitution, deletion or addition of one or several amino acid residues from the amino acid sequence of SEQ ID 2 Protein acts like 2

SEQ ID 1 in the Sequence Listing consists of 501 base pairs. Its leading frame is 1-498 nucleotides from the 5'-end.

Expression vectors and cell lines (eg, Escherichia coli and yeast expression systems, etc.) comprising the genes of the invention are within the scope of the invention.

Example 1

Cyclic Exchange of Recombinant Humans TRAIL Construction and Expression of Coding Genes

Gene sequences encoding amino acid residues at positions 121-281 of human TRAIL can be obtained from the human spleen cDNA library. Genes encoding amino acid residues at positions 135-281 of human TRAIL can be made by a general PCR method using the human TRAIL gene as a template. The DNA sequence encoding amino acid residues 122-135 of TRAIL was linked by the PCR method to the 3'-end of the DNA sequence encoding the amino acid residue at position 135-281 of TRAIL. A DNA sequence encoding five glycine residues was inserted between the DNA sequence encoding amino acid residues at positions 135-281 of TRAIL and the DNA sequence encoding amino acid residues at positions 122-135 of TRAIL. The flexibility of glycine allows for proper protein folding. The gene encoding the CPT thus obtained is linked to the vector plasmid pet28b (or other vector plasmid) using Ncol and BamHI to become an expression plasmid. This DNA is correctly identified through sequencing.

Expression plasmids were transformed with E. coli variant BL21 (DE3). Transformed E. coli was mixed in 10 ml of LB liquid medium containing 20 μg / ml kanamycin. Cells were incubated for 12 hours at 37 ° C. in a shaker. 10 ml of the culture solution was mixed with 1 L of LB liquid medium containing 20 μg / ml of kanamycin and further culture was performed. When OD ₆₀₀ reached 0.6, 0.2 ml of 1 M IPTG was added to 1 L of culture to induce protein expression. Cells were harvested by centrifugation after induction for 3 hours. The pellets were stored in 100 ml buffer containing 100 ml Tris (pH 7.9) and 150 mM sodium chloride.

Cells were lysed at 4 ° C. and separated at 15,000 rpm using a Beckman JA20 rotor. Since the expressed protein can bind to the metal chelate resin, it can be purified by metal chelate chromatography. After centrifugation, the supernatant was move to a fixed Ni ^{2 +} chelate chromatography column. This column was washed with a buffer containing 50 mM Tris (pH 7.9), 0.5 M sodium chloride and 50 mM imidazole to remove contaminating proteins contained therein. The bound protein was isolated in a buffer containing 50 mM Tris (pH 7.9), 0.5 M sodium chloride and 200 mM imidazole. The isolated protein was dialyzed against PBS buffer.

Finally, the protein was purified by gel permeation column Superdex 200 (Pharmacia) embedded in an ion exchange column and AKTA HPLC system (Pharmacia). Analysis of the protein means that the protein thus obtained has the amino acid sequence of SEQ ID 2, the anticancer drug of the present invention. The final product was a white powdered powder and dissolved in water.

Example 2

Tetrazolium  Reduction method ( MTT To determine the toxic effect of the drug of the present invention on cancer cells

Reagent: RPMI 1640 is obtained from GIBCO. MTT is obtained from Bebco. Bovine serum albumin is purchased from Hangzhou Shijiking Biotech Co., Ltd. (China).

Cell Features: COLO 205 (human colon cancer cells), NCI-H460 (human lung cancer cells), RPMI 8226 (human multiple myeloma cells) and U251 (human brain glioma cells) are obtained from the US ATCC. HL-60 (human promyelocytic leukemia cells), MDA-MB-231, MDA-MB-435 (human breast cancer cells), SCLC (human small cell lung cancer cells), H125 (human lung cancer cells) and PC-3 (human pancreatic cancer cell).

Growing tumor cells were harvested to prepare a cell suspension (1 × 10 ⁴ / ml) with RPMI 1640 medium containing 10% bovine serum albumin. Cell suspensions are inoculated in 96 well plates, 100 μL per well (containing 1000 tumor cells). This plate was incubated for 24 hours in a 5% carbon dioxide incubator at 37 ° C. and then drug added.

Blank and positive controls with 5-fluorouracil or adriamycin were used in this experiment. The CPT sample has 5 concentrations and 3 parallel wells for each concentration. Cells were incubated in incubator for 4 days at 37 ° C. with 5% carbon dioxide. The culture medium was then removed and 100 μL of MTT solution (0.4 mg / ml of RPMI 1640) added to each well. This plate was incubated at 37 ° C. for an additional 4 hours.

Supernatant was discarded. 150 μL DMSO was added to each well to dissolve the formazan granules. After gentle shaking, the OD value is measured by the BIORAD 550 Enzyme Analyzer with a sensing wavelength of 540 nm and a reference wavelength of 450 nm.

Dose-response curves consisted of cell inhibition rates and various concentrations of drug, with half inhibitory concentrations (IC ₅₀ ) calculated. The results are shown in Table 1. CPT has a very potent killing effect on human tumor cells U251, COLO205, NCI-H460 and MDA-MB-435 with IC ₅₀ <0.01 μg / ml. In addition, CPT has a very potent killing effect on HL-60, MDA-MB-231, PC-3 and HL125 cells, IC ₅₀ <1 μg / ml. CPT also has a slightly inhibitory effect on the growth of SCLC cells. CPT activity, which induces apoptosis of cancer cells, is 6-11 times stronger than that of wild type TRAIL.

Killing Effect of CPT on Human Tumor Cell Types (× ± SD) Tumor cell types IC ₅₀ (μg / ml) CPT Fu Adr U251 <0.001 0.981 ± 0.077 COLO205 0.006 ± 0 0.826 ± 0.051 MDA-MB-435 0.008 ± 0.002 0.199 ± 0.138 HL-60 0.014 ± 0.013 0.006 ± 0 NCI-H460 0.002 ± 0.001 0.566 ± 0.016 MDA-MB-231 0.043 ± 0.0131 0.318 ± 0.055 PC-3 0.067 ± 0.002 4.928 ± 0.753 H125 0.085 ± 0.006 0.138 ± 0.031 RPMI8226 0.824 ± 0.093 <0.1 SCLC 10 ± 0 0.617 ± 0.0257

Note: FU refers to the 5-fluorouracil control and Adr refers to the adriamycin control.

Example 3

Inhibitory effect of the drug according to the invention on the growth of human cancer xenografted in nude mice.

In this example, the animals used are BALB / c-nu nude mice, 6-8 weeks old. All animals used in each experiment are homosexual.

Tumor Cell Types: Human Brain Glioma U251, Human Lung Cancer Cell Line NCI-H460, Human Colon Cancer Cell Line COLO205 is inoculated subcutaneously from nude in vitro from the culture, and the tumor is maintained in secondary culture.

Experimental Procedures: Grown nude mice with grown tumors were selected to dislocate and kill the neck vertebrae. The tumor was removed by sterile treatment and cut into pieces having a diameter of 2-3 mm using a surgical scalpel. These pieces were inoculated subcutaneously with a needle in the nucleus of the mouse. Tumors may be found in the mesenchymal fluid of animals after about 7-10 days. Tumor length and width were measured by vernier calipers. Animals are classified according to their tumor size. Each group contained 6-8 animals.

Tumor length and width and mouse weight were measured twice a week. Tumor volume (TV) and relative tumor volume (RTV) were calculated. A graph showing the change in tumor size was created. At the end of the experiment, tumors were removed and the dead mice weighed to calculate the inhibition rate of the drug in tumor growth.

Equation for calculating tumor volume (TV): length × width ² ÷ 2.

Formula for calculating relative tumor volume (RTV): Vt / Vo (Vo is the TV value measured at the start of inoculation and Vt is the TV value measured thereafter).

T-tests were used to determine statistical differences in tumor weight, tumor volume and RTV between different groups of animals. The indicator used to assess anticancer activity is the relative tumorigenic rate T / C (%).

T / C (%) = of drug recipient RTV (T) × 100

            Voice Control RTV (C)

Evaluation criteria to know efficacy:

T / C (%)> 60 is invalid.

T / C (%) ≤60 and P <0.05 after statistical analysis was invalidated.

1. Inhibitory Effect on Human Brain Glioma U251 .

Carmustine (Lenmin Pharmaceuticals, China's Tanjin Amino Acids) is used as a positive control, administered at 40 mg / kg once via intraperitoneal injection. CPT was administered to four groups via intraperitoneal injection with doses of 0.6 mg / kg, 1.7 mg / kg, 5.0 mg / kg and 15.0 mg / kg, respectively. Treatment included 10 injections.

Experimental results can be seen in Tables 1 and 2 and FIGS. 1 and 2. It can be seen that CPT shows an important inhibitory effect on the growth of transplanted tumors (human brain glioma U251) in nude mice. Tumor inhibition rates for the four treatment groups calculated by body weight were 30.5%, 44.5%, 64.8% and 87.5%, respectively. Compared with the control group, the tumor weight of the treated group showed a significant or very significant statistical difference. The anticancer effect of CPT is significantly dependent on the dose. The relative tumor growth rate T / C (%) values for the 5.0 mg / kg and 15 mg / kg groups were less than 60. Relative tumor volume (RTV) for the 15 mg / kg group showed statistically significant differences when compared to that of the control group.

Inhibitory Effect of CPT on the Growth of Human Glioma U251 (× ± SD) group Animal count Weight (g) Tumor weight (g) % Inhibition start End start End Negative control 8 8 21.9 ± 1.1 22.3 ± 2.6 1.28 ± 0.50 Carmustine Control 8 7 22.8 ± 0.7 19.4 ± 2.1 0.09 ± 0.06 ** 93.0 CPT 0.6mg / kg 8 8 21.0 ± 0.8 21.0 ± 1.4 0.89 ± 0.49 30.5 CPT 1.7mg / kg 8 8 21.6 ± 1.2 21.5 ± 2.0 0.71 ± 0.22 * 44.5 CPT 5.0mg / kg 8 8 22.0 ± 1.1 21.9 ± 3.2 0.45 ± 0.26 ** 64.8 CPT 15mg / kg 8 8 21.6 ± 1.1 21.7 ± 1.7 0.16 ± 0.15 ** 87.5

Inhibitory Effect of CPT on the Growth of Human Glioma U251 (× ± SD) group Tumor volume (mm ³ ) RTV T / C (%) start End Negative Control 165 ± 72 1238 ± 244 3.34 ± 2.28 Carmustine Control 153 ± 35 109 ± 78 ** 0.54 ± 0.29 ** 16.0 CPT 0.6mg / kg 141 ± 40 961 ± 582 2.77 ± 1.97 82.9 CPT 1.7mg / kg 131 ± 34 588 ± 215 ** 2.12 ± 1.29 63.5 CPT 5.0mg / kg 133 ± 57 510 ± 333 ** 1.32 ± 1.24 ** 39.5 CPT 15mg / kg 148 ± 58 261 ± 238 ** 0.71 ± 0.57 ** 21.3

* P <0.05 ** P <0.01

2. Inhibitory Effect on Human Lung Cancer NCI - H460

Cyclopamide (Shanghai Huanglian Pharmaceutical Co., Ltd.) was used as a positive control, administered by intraperitoneal injection, with a single dose of 100 mg / kg. The booster was 2 weeks later and the dose was 80 mg / kg. CPT was given to the three treatment groups in the amounts of 1.7 mg / kg, 5.0 mg / kg and 15.0 mg / kg, respectively, and was administered by intraperitoneal injection once a day. Treatment included a total of 10 injections.

The results of the experiment are shown in Tables 4 and 5 and FIGS. 3 and 5. It was found that CPT showed a significant inhibitory effect on the growth of transplanted tumors (human lung cancer NCI-H460) in nude mice. Tumor inhibition rates for the three treatment groups, calculated by body weight, were 52.2%, 74.5% and 87.0%, respectively. Compared with the control group, the tumor weight of the treated group showed a significant or very significant statistical difference. Relative tumor growth rate (%) values for the 5.0 mg / kg and 15 mg / kg groups are less than 60. Relative tumor volume (RTV) was statistically significant compared to the control.

Inhibitory Effect of CPT on the Growth of Human Lung Cancer NCI-H460 group Animal count Weight (g) Tumor Weight (g) % Inhibition start End start End Negative Control 8 7 21.4 ± 0.9 19.3 ± 1.0 1.61 ± 0.65 Cyclophosphamide control 8 7 20.0 ± 1.3 16.1 ± 2.2 0.96 ± 0.35 ** 40.4 CPT 1.7mg / kg 8 7 20.1 ± 1.9 19.8 ± 1.6 0.77 ± 0.19 ** 52.2 CPT 5.0mg / kg 8 8 20.4 ± 1.9 18.5 ± 2.5 0.41 ± 0.28 ** 74.5 CPT 15mg / kg 8 8 20.9 ± 1.1 21.1 ± 1.7 0.21 ± 0.10 ** 87.0

Inhibitory Effect of CPT on the Growth of Human Lung Cancer NCI-H460 group Tumor volume (mm ³ ) RTV T / C (%) start End Negative control 169 ± 58 1874 ± 637 5.98 ± 4.05 Cyclophosphamide control 179 ± 52 1121 ± 434 3.67 ± 2.15 61.4 CPT 1.7mg / kg 200 ± 103 706 ± 170 * 2.27 ± 1.13 * 38.0 CPT 5.0mg / kg 187 ± 51 469 ± 359 ** 1.49 ± 0.76 ** 24.9 CPT 15mg / kg 192 ± 63 189 ± 102 ** 0.79 ± 0.25 ** 13.2

 * P <0.05 ** compared to P <0.01 negative control

The results of histopathology tests can be seen in Figure 4A to 4F, Figures 4A, 4C and 4E are negative controls. 4A shows that tumor tissue spreads to resemble a bundle. In the middle of the tumor is a large soluble necrosis. Tumors are surrounded by connective tissue membranes and rarely penetrated by lymphocytes. 4C shows that tumor cells are arranged in a structure such as a solid bundle and cord. Tumor tissue is rich in capillaries and rarely penetrated by lymphocytes. 4E shows that tumor cells are rounded or polygonal, have large nuclei and little cytoplasm. There are two or three nuclei that are loose in chromatin and distinct in nuclei and show evidence of various mitosis. 4b, 4d and 4f are groups treated with 15 mg / kg CPT. 4B shows that the tumor tissue is a solid bundle and is a central paper-like necrosis. Tumors have abundantly expanded connective tissue, and thin connective tissue surrounds the tumor. Tumors are rarely infiltrated by lymphocytes. 4D shows that tumor cells are arranged in a cord-like structure. Tumor tissue rarely has capillaries and has gangrene in the center. Necrosis is surrounded by connective tissue envelope and infiltrated by some lymphocytes. 4F shows that the tumor cells are round or polygonal, large in nucleus and contain little cytoplasm. Chromatin is loose and there are signs of mitosis. Tumors rarely have capillaries and have gangrene with small focal points such as multiple spots.

It is apparent that the drug according to the present invention has a strong inhibitory effect on human lung cancer NCI-H460 cells.

3. Inhibitory Effects on the Growth of Human Colon Cancer COLO 205

Injectable hydroxycamtothecin (trade name: Wuhan Rishizen Si Shi, Huangshi Rishizen Pharmaceutical Group, China) was used as a positive control and was administered once daily by intraperitoneal injection in an amount of 1 mg / kg once . Dosing was terminated after 15 days of continuous dosing. It was observed that tumor growth was significant; Therefore, an additional week of hydroxycamptothecin was made via intraperitoneal injection in an amount of 100 mg / kg after 8 days. One dose of hydroxycamptothecin at 80 mg / kg was additionally made after two weeks. CPT was given to doses of 1.7 mg / kg, 5.0 mg / kg and 15.0 mg / kg 3, respectively, intraperitoneally once daily. Treatment included a total of 15 injections.

Experimental results are shown in Tables 6 and 7, Figures 6 and 7a to 7e, Figure 7a is a negative control, Figure 7b is for the treatment group by 15.0mg / kg CPT, Figure 7c is a treatment group by 5.0mg / kg CPT FIG. 7D is for the treatment group with 1.7 mg / kg CPT and FIG. 7E is for the hydroxycamptothecin positive control. The results show that CPT shows an important inhibitory effect on the growth of transplanted tumors (human colon cancer COLO 205) in nude mice. After 5 days of treatment, the tumor size was significantly reduced in the large CPT treatment group. Some tumors even disappeared with increased doses. Observation of these mice was performed 4 weeks after discontinuation and no tumor was found in one third of the animals. Anatomical analysis of these mice revealed only traces of inoculation in the skin. Tumor inhibition rates for the three treatment groups, calculated by body weight, were 79.6%, 90.8% and 97.4%, respectively. Compared with the control group, the tumor weight of all treatment groups showed a significant or very significant statistical difference. The relative tumor growth rate T / C (%) values for all treatment groups were less than 60. Relative tumor volume (RTV) is significantly different compared to the dimensions of the control group. In particular, the T / C (%) value for the treatment group with large amounts of CPT is less than 10, indicating that CPT plays a large role in this type of tumor.

Inhibitory Effect of CPT on the Growth of Human Colon Cancer COLO 205 group Animal count Weight (g) Tumor Weight (g) % Inhibition start End start End Negative control 8 8 19.5 ± 0.8 21.9 ± 1.9 1.96 ± 0.73 Hydroxy camptothecin control 8 8 20.4 ± 0.9 21.0 ± 2.5 0.66 ± 0.26 ** 66.3 CPT 1.7mg / kg 8 8 20.6 ± 1.1 20.6 ± 3.0 0.40 ± 0.33 ** 79.6 CPT 5.0mg / kg 8 7 20.2 ± 0.8 21.4 ± 1.2 0.18 ± 0.15 ** 90.9 CPT 15mg / kg 8 8 20.0 ± 0.5 21.1 ± 2.4 0.05 ± 0.05 ** 97.4

Inhibitory Effect of CPT on the Growth of Human Colon Cancer COLO 205 group Tumor volume (mm ³ ) RTV T / C (%) start End Negative control 151 ± 79 2532 ± 1190 8.30 ± 5.53 Hydroxy camptothecin control 150 ± 34 845 ± 66 ** 3.92 ± 1.91 * 47.2 CPT 1.7mg / kg 205 ± 47 582 ± 408 * 1.62 ± 0.62 ** 19.5 CPT 5.0mg / kg 180 ± 63 314 ± 175 ** 0.91 ± 0.35 ** 11.9 CPT 15mg / kg 192 ± 81 52 ± 53 ** 0.33 ± 0.27 ** 4.0

  * P <0.05 ** compared to P <0.01 negative control

Industrial availability

Tetrazolium reduction MTT was used to determine the anticancer effect of the drug. Dose-response curves consisted of the percentage inhibition of cells and the various concentrations of drug, with half the inhibition concentration (IC ₅₀ ) calculated. Observations on many human tumor cell lines demonstrate that CPT formation has a very large inhibitory effect on the growth of tumor cells. CPT kills more than 10 tumor cells, including human lung cancer, colon cancer, breast cancer, gastric cancer, pancreatic cancer, glioma, hematopoietic cancer, bladder cancer, prostate cancer, colon cancer, cervical cancer, and brain tumor cells. The anti-inhibitory concentration (IC ₅₀ ) of CPT for U251 (human brain glioma cells), COLO 205 (human colon cancer cells), NCI-H460 (human lung cancer cells), MDA-MB-435 (human breast cancer cells) was 0.01 μg. less than / ml IC ₅₀ values for CPT of HL-60 (human promyelocytic leukemia cells), MDA-MB-231 (human breast cancer cells), PC-3 (human pancreatic cancer cells) and H125 (human lung cancer cells) are less than 0.1 μg / ml small. IC _{50 of} CPT for RPMI 8226 (human multiple myeloma cells) The value is less than 1 μg / ml. CPT also shows an inhibitory effect on the growth of SCLC (human small cell lung cancer).

In vivo experiments showed that intraperitoneal administration of CPT at a dose of 15 mg / kg per day for 10 to 15 days continued on growth of tumor cells including human colon cancer COLO 205, human lung cancer NCI-H460 and human glioma U251. It shows a very serious inhibitory effect.

CPT can significantly reduce the growth rate of tumors in animals when compared to negative controls that do not take any drugs, and large doses in the early stages of growth of transplanted tumors reduce or even reduce tumor volume. It may be gone. The effect of CPT is clearly dose dependent. In addition, efficacy is somewhat related to the process. In nude mouse models implanted with human colon cancer COLO 205, the size of the tumor was observed to be much smaller than that before the administration even after 4 weeks of discontinuation, where mice continued to receive CPT for 15 days at a dose of 15 mg / kg per day. Administered. The presence of tumors could not be identified in the inoculation area in one third of the animals, only indications caused by inoculation were observed during anatomical analysis.

All data show that administration of CPT has an important inhibitory effect on the growth of human tumors transplanted into nude mice. In the case of U251, the inhibition rate of 15 mg / kg CPT based on tumor weight was 87.5% and 87.7%, respectively, compared to the control group. For NCI-H460, inhibition rates were 87.0% and 88.8%, respectively. In the case of COLO 205, the inhibition rates were 97.4% and 97.8%, respectively, which showed a very significant difference statistically. Three models were used to run the experiments in this invention. The results show that the relative tumor area (RTV) of the three animal models is statistically significantly different from the negative control group, which continued to receive CPT at 5.0 mg / kg for 10-15 days. The data for the CPT treated group at 15 mg / kg are very different from the negative control, where all T / C (%) values are below 25 and even below 10. This means that CPT has high anticancer activity and that the experiment is repeatable.

CPT showed no obvious side effects or toxic effects on animals. There were no significant differences in the survival status of the mice, such as body weight, and the treatment group's viability compared to the negative control, except that the tumors were smaller in the treated mice. This suggests that CPT has a very selective inhibitory effect on tumor cells and that CPT does not induce cellular apoptosis of normal tissues. Therefore, CPT can be used as an effective and safe drug for treating cancer. CPT is theoretically all industrially important and has bright prospects in the market.

<110> BEIJING SUNBIO BIOTECH CO., LTD. <120> A RECOMBINANT PROTEIN WITH CANCER SUPPRESSION ACTION, ITS          ENCODING GENE AND USE <130> PI-6K1042CN <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 501 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 acattgtctt ctccaaactc caagaatgaa aaggctctgg gccgcaaaat aaactcctgg 60 gaatcatcaa ggagtgggca ttcattcctg agcaacttgc acttgaggaa tggtgaactg 120 gtcatccatg aaaaagggtt ttactacatc tattcccaaa catactttcg atttcaggag 180 gaaataaaag aaaacacaaa gaacgacaaa caaatggtcc aatatattta caaatacaca 240 agttatcctg accctatatt gttgatgaaa agtgctagaa atagttgttg gtctaaagat 300 gcagaatatg gactctattc catctatcaa gggggaatat ttgagcttaa ggaaaatgac 360 agaatttttg tttctgtaac aaatgagcac ttgatagaca tggaccatga agccagtttt 420 tttggggcct ttttagttgg cggtggtggt ggtggtgtag cagctcacat aactgggacc 480 agaggaagaa gcaacacata a 501 <210> 2 <211> 166 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys   1 5 10 15 Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn              20 25 30 Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr          35 40 45 Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu      50 55 60 Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr  65 70 75 80 Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys                  85 90 95 Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly             100 105 110 Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn         115 120 125 Glu His Leu Ile Asp Met Asp His Glu Ala Ser Phe Ply Gly Ala Phe     130 135 140 Leu Val Gly Gly Gly Gly Gly Gly Val Ala Ala His Ile Thr Gly Thr 145 150 155 160 Arg Gly Arg Ser Asn Thr                 165

Claims (14)

1) a protein having the amino acid sequence of SEQ ID 2 in the sequence list 2) a protein derived from SEQ ID 2 and having at least 90% homology with SEQ ID 2 and having the same activity, 3) A protein derived from SEQ ID 2, obtained by substituting, deleting or adding one or several amino acid residues from the amino acid sequence of SEQ ID 2, and having an anticancer effect as one selected from proteins having the same activity as SEQ ID 2; Branched recombinant protein. The method of claim 1, Wherein said protein is SEQ ID 2 in the sequence listing. 1) SEQ ID 1 of the sequence list 2) a polynucleotide capable of encoding the amino acid sequence of SEQ ID 2 in the sequence listing 3) a DNA sequence encoding a protein having at least 90% sequence homology with a DNA sequence defined by SEQ ID 1 of the Sequence Listing and having the same activity as the protein encoded by SEQ ID 1 4) A DNA sequence encoding a protein derived from SEQ ID 2, wherein the protein derived from SEQ ID 2 is obtained by substitution, deletion or addition of one or several amino acid residues from the amino acid sequence of SEQ ID 2 A gene selected from among proteins having the same activity as ID 2, which encodes a recombinant protein having an anticancer effect. The method of claim 3, The gene is SEQ ID 1 in the sequence list. A cancer therapeutic agent comprising the recombinant protein of claim 1 or 2 as an active ingredient. An expression vector comprising the gene of claim 3 or 4. A cell line comprising the gene of claim 3 or 4. Use of a recombinant protein for the preparation of a medicament for the treatment of the cancer of claim 1. The method of claim 8, Cancer is selected from colon cancer, lung cancer, various myeloma, brain glioma, leukemia, breast cancer, small cell lung cancer and pancreatic cancer. The method of claim 9, The cancer is characterized in that the various myeloma. The method of claim 9, The cancer is characterized in that the leukemia. The method of claim 9, Cancer is a brain glioma. The method of claim 9, The cancer is lung cancer. The method of claim 9, The cancer is colon cancer.

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