RU2333221C2 - Recombinant albumen with anti-cancer effect, gene coding it, and application thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: according to the invention, the recombinant albumen with anti-cancer effect is selected out of the group including 1) albumen with amino acid sequence presented in SEQ ID N0:2 indicated in the sequence list; 2) albumen with amino acid sequence homologous to the sequence presented in SEQ ID NO:2 by more than 90% and featuring the same effect as the albumen 1); 3) albumen obtained by replacement, deletion or addition of one or more residua to the amino acid sequence presented in SEQ ID NO:2 and featuring the same effect as the albumen 1). The invention also claims a gene coding the described recombinant albumen and its variants, expression vector containing the said gene, and cell line containing the said gene and intended for expression of the recombinant albumen. The recombinant albumen can be applied in obtaining cancer treatment medication. A medication including the aforesaid recombinant albumen as active component has strong selective effect on tumour cells and does not cause apoptosis of normal tissue cells.

EFFECT: developed substance for obtaining cancer treating medication.

14 cl, 16 dwg, 7 tbl, 3 ex

Description

FIELD OF THE INVENTION

The present invention relates to the field of genetic engineering and pharmacology. The present invention relates to a recombinant protein encoding its gene and a genetically engineered drug containing said recombinant protein as an active ingredient. In particular, the present invention relates to a recombinant protein having an anti-cancer effect, an encoding gene thereof, and a cancer medicament containing said recombinant protein as an active ingredient.

State of the art

As mammals evolved, a number of signaling mechanisms for apoptosis gradually emerged that could induce programmed death of individual cells. The underlying theory is that lethal cell ligands interact with death receptors on cell surfaces, which induces cell apoptosis. Such beneficial apoptosis plays an important physiological role in the removal of activated lymphocytes at the end of the immune response and in the destruction of virus-infected cells and oncogenic cells. Examples of this interaction include the interaction between TNF and the TNFR receptor and the interaction between FasL and the Fas / Apo1 / CD95 receptor.

Activated death receptors are directly involved in cascading cell apoptosis reactions. They can induce apoptosis of various cancer cells and are likely anti-cancer factors. Although TNF and FasL can induce apoptosis of cancer cells, they cause severe toxic and side effects when used in anti-cancer therapy. TNF injection can lead to lethal inflammatory reactions similar to septic shock. This reaction is mainly mediated by NF-κB, a pre-transcription factor that is localized in vascular endothelial cells and macrophages and which is activated by TNF. An anti-Fas antibody can induce Fas-dependent apoptosis of cells in the liver tissue and cause fatal liver damage.

In 1995, Wiley et al. found a TNF-related apoptosis-inducing ligand (TRAIL), based on the identity of its sequence with TNF and FasL. TRAIL can induce apoptosis by interacting with the death receptor DR4 or DR5. Unlike TNF and FasL, TRAIL mRNA is constitutively expressed in many normal human tissues. This indicates a physiological mechanism by which TRAIL induces apoptosis of cancer cells without affecting normal cells. These mechanisms include the expression of inhibitory receptors, the ability to compete with DR4 and DR5 for binding to ligands, and the ability of TRAIL to interact with the three receptors DcR1, DcR2, and DPG. Thus, the toxicity of TRAIL is less than the toxicity of TNF and FasL.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a recombinant protein having an anti-cancer effect and a medicament for the effective treatment of cancer, which contains this recombinant protein as an active ingredient.

A recombinant protein having an anti-cancer effect of the present invention is a protein selected from the group consisting of:

1) a protein with the amino acid sequence of SEQ ID NO: 2 shown in the list of sequences;

2) a protein based on SEQ ID NO: 2, the sequence of which is homologous to the sequence of SEQ ID NO 2 by more than 90% and which has the same activity as SEQ ID NO: 2;

3) a protein based on SEQ ID NO: 2, which is obtained by the addition or deletion of 15 or less amino acid residues at the N-terminus of the amino acid sequence of SEQ ID NO: 2 and which has the same activity as SEQ ID NO: 2;

4) a protein based on SEQ ID NO: 2, which is obtained by the addition or deletion of 15 or less amino acid residues at the C-terminus of the amino acid sequence of SEQ ID NO: 2 and which has the same activity as SEQ ID NO: 2;

5) a protein based on SEQ ID NO: 2, which is obtained by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 2 and which has the same activity as SEQ ID NO: 2.

The protein SEQ ID NO: 2 shown in the sequence list is cyclically permuted human TRAIL (CPT), which consists of 166 amino acid residues.

The active ingredient of a cancer medicine obtained in the present invention (CPT) is one of the aforementioned proteins.

If desired, one or more pharmaceutically acceptable carriers may be added to the above medicament. The carrier includes a diluent, an excipient, a filler, a binder, a moisturizing agent, a disintegrating agent, an absorption enhancer, a surfactant, an adsorbent carrier, a lubricant commonly used in the pharmaceutical field. If necessary, flavoring, sweetening, and the like may also be added.

The drug of the present invention can be prepared in various forms, including injection, tablets, powders, granules, capsules, oral liquid, ointment and cream, etc. Any of the aforementioned dosage forms can be obtained by conventional methods in the pharmaceutical field.

The coding sequence of the active ingredient (protein) of a cancer treatment drug (CPT) of the present invention is a sequence selected from the group consisting of:

1) SEQ ID NO: 1 shown in the list of sequences;

2) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 shown in the list of sequences;

3) a DNA sequence that is more than 90% homologous to the DNA sequence of SEQ ID NO: 1 shown in the sequence list and which encodes a protein with the same activity as the protein encoded by SEQ ID NO: 1;

4) a DNA sequence encoding a protein based on SEQ ID NO: 2, where the specified protein based on SEQ ID NO: 2, obtained by the addition or deletion of 15 or less amino acid residues at the N-end of the amino acid sequence of SEQ ID NO: 2 and has such the same activity as SEQ ID NO: 2;

5) a DNA sequence encoding a protein based on SEQ ID NO: 2, wherein said protein based on SEQ ID NO: 2 is obtained by the addition or deletion of 15 or less amino acid residues at the C-terminus of the amino acid sequence of SEQ ID NO: 2 and has the same activity as SEQ ID NO: 2;

6) a DNA sequence encoding a protein based on SEQ ID NO: 2, wherein said protein based on SEQ ID NO: 2 is obtained by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 2 and has the same activity, like SEQ ID NO: 2.

The sequence of SEQ ID NO: 1 in the list of sequences consists of 501 base pairs. Its reading frame is in the region of nucleotides 1-498 from the 5'-end.

Expression vectors and cell lines (such as Escherichia coli and yeast expression systems, etc.) containing the gene of the invention are within the scope of the present invention.

A brief description of the graphic material

In FIG. 1 shows the volume curves of human glioma U251 tumors under various treatment conditions.

In FIG. Figure 2 shows the sizes of human glioma U251 tumors under various treatment conditions.

In FIG. Figure 3 shows the volume curves of human lung cancer tumors NCI-H460 under various treatment conditions.

In FIG. 4-1 - FIG. Figures 4-6 show histopathological sections of human lung cancer NCI-H460 under various treatment conditions (HE-staining x 100).

In FIG. Figure 5 shows the size of human lung tumors NCI-H460 under various treatment conditions.

In FIG. Figure 6 shows the volume curves of human colorectal cancer tumors COLO 205 under various treatment conditions.

In FIG. 7-1 - FIG. Figures 7-5 show Balb / c-nu nude mice that were inoculated with human colorectal cancer COLO 205, followed by various treatment regimens.

BEST MODES FOR CARRYING OUT THE INVENTION

Example 1

Construction and expression of the coding gene of recombinant cyclically permuted human TRAIL

The gene sequence encoding the amino acid residues at positions 121-281 of the human TRAIL was obtained from the human spleen cDNA library. The gene encoding the amino acid residues at positions 135-281 of the human TRAIL was obtained by the usual PCR method using the human TRAIL gene as a template. Then the DNA sequence encoding the amino acid residues 122-135 TRAIL, was associated with the 3'-end of the DNA sequence encoding the amino acid residues at positions 135-281 TRAIL, PCR method. The DNA sequence encoding the five glycine residues was inserted between the DNA sequence encoding the amino acid residues 135-281 TRAIL and the DNA sequence encoding the amino acid residues 122-135 TRAIL. Glycine motility may facilitate proper protein folding. The CPT encoding gene thus obtained was ligated into the pet28b vector plasmid (or other vector plasmid) using NcoI and BamHI to obtain an expression plasmid. By sequencing, it was confirmed that the DNA sequence is correct.

The expression plasmid was transformed into E. coli strain BL21 (DE3). Transformed E. coli was inoculated in 10 ml LB liquid medium containing 20 μg / ml kanamycin. Cells were incubated on a shaker at 37 ° C for 12 hours. Then, 10 ml of the culture was inoculated in 1 L of LB liquid medium containing 20 μg / ml kanamycin, and cultivation was continued. Upon reaching OD _{600 of} 0.6, 0.2 ml of 1 M IPTG in 1 L culture was added to induce protein expression. Cells were harvested by centrifugation three hours after induction. The precipitate was suspended in 100 ml of buffer containing 100 mM Tris (pH 7.9) and 150 mM NaCl.

Cells were lysed by sonication at 4 ° C and centrifuged at 15,000 rpm using a Beckman JA20 rotor. Since the expressed protein is capable of binding to metal chelating polymers, it can be purified using metal chelate chromatography. After centrifugation, the supernatant was applied using a pump to a chromatographic column with immobilized Ni ²⁺ chelate. The column was washed with a buffer containing 50 mM Tris (pH 7.9), 0.05 M NaCl and 50 mM imidazole to remove the contaminating proteins contained therein. The bound protein was then eluted with a buffer containing 50 mM Tris (pH 7.9), 0.5 M NaCl and 200 mM imidazole. The eluted protein was dialyzed against PBS buffer.

Finally, the protein was purified using an ion exchange column and a Superdex 200 gel filtration column (Pharmacia), placed in an AKTA LC system (Pharmacia). Analysis of the protein showed that the protein thus obtained has the amino acid sequence of SEQ ID NO: 2, which is the anticancer drug of the invention. The final product was a white powder and was water soluble.

Example 2

Determination of the cytotoxic effect of a drug of the present invention on cancer cells using a tetrazolium (MTT) recovery method

Reagents: RPMI 1640 obtained from Gibco; MTT from Bebco. Fetal calf serum purchased from Hangzhou SiJiQing Bioengineering Material Co. Ltd. (P.R. China).

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

Well-growing tumor cells were harvested to prepare a cell suspension (1x10 ⁴ / ml) using RPMI 1640 medium containing 10% fetal calf serum. The cell suspension was inoculated into a 96-well plate, 100 μl (containing 1000 tumor cells) per well. The tablet was cultured in a thermostat with 5% CO ₂ at 37 ° C for 24 hours, followed by the addition of the drug.

Blind control and positive control with 5-fluorouracil or adriamycin were used in this experiment. CPT samples were obtained in five concentrations; three parallel wells were used for each concentration. Cells were cultured in an incubator with 5% CO ₂ at 37 ° C for 4 days. The culture medium was then removed and 100 μl of MTT solution (0.4 mg / ml in RPMI 1640) was added to each well. The plate was incubated at 37 ° C for another four hours.

The supernatant was removed. 150 μl of DMSO was added to each well to solubilize Fomazan granules. After careful shaking, the OD values were measured using a BIORAD 550 enzyme analyzer with a determination wavelength of 540 nm and a reference wavelength of 450 nm.

Dose response curves were plotted with cell inhibition intensities and various drug concentrations, and 50% inhibition concentrations were calculated (IC ₅₀ ). The results are shown in Table 1. The graph shows that CPT has an extremely strong cytolysis effect on human tumor cells U251, COLO 205, NCI-H460, MDA-MB-435, where IC ₅₀ <0.01 μg / ml. CPT also has a very strong cytolysis effect on HL-60, MDA-MB-231, PC-3 and HL125 cells, where IC ₅₀ <0.1 μg / ml. CPT has a good effect on RPMI 8226 cells, where IC ₅₀ <1 μg / ml. Some of its inhibitory effects on SCLC cell growth have also been shown. The activity of CPT with respect to the induction of apoptosis of cancer cells is six to eleven times stronger than the activity of wild-type TRAIL.

Table 1
The cytolysis effect of CPT on the human tumor cell line (x ± SD) Tumor cell line IC ₅₀ (μg / ml) CPT Fu Adr U251 <0.001 0.981 ± 0.077 COLO 205 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 RPMI 8226 0.824 ± 0.093 <0.1 SCLC 10 ± 0 0.617 ± 0.0257 Note. FU is 5-fluorouracil (control);
Adr stands for adriamycin (control).

Example 3

The inhibitory effect of the drug of the present invention on the growth of human cancer xenograft in nude mice

In this example, the animals used were Balb / c-nu nude mice, 6-8 weeks old. In each experiment, all animals were of the same sex.

Tumor cell lines: human brain glioma U251 cells, NCI-H460 human lung cancer cell line, human colon cancer cell line COLO 205 were inoculated subcutaneously from in vitro culture in nude mice, tumors were subcultured and stored.

Experimental procedures: well-grown “naked” mice with a sufficiently grown tumor were selected and killed by displacement of the cervical vertebra. Tumors were extracted aseptically and cut with a scalpel into pieces with a diameter of 2-3 mm. The pieces were inoculated into the axillary fossa of the animals subcutaneously with a needle. After approximately 7-10 days, tumors could be detected in the axillary fossa of these animals. The length and width of the tumors were measured with a caliper. Then these animals were grouped based on the size of the tumors. Each group included seven to eight animals.

The length, tumor width, and body weight of the mice were measured twice a week. Tumor volume (TV) and relative tumor volume (RTV) were calculated. A graph was constructed showing changes in tumor size. At the end of this experiment, tumors were recovered and weighed after killing the mice, and the inhibition rates of the test drug with respect to tumor growth were calculated.

The formula for calculating tumor volume (TV) was as follows: length × width ² - 2.

The formula for calculating the relative tumor volume (RTV) was as follows: Vt / Vo (where Vo is equal to the value of TV measured at the beginning of administration, Vt is equal to the value of TV measured thereafter).

The T-test was used to determine the statistical difference in tumor masses, tumor volumes and RTV, etc. between different groups of animals. The index used to evaluate antitumor activity is the relative growth rate T / C (%), the formula for which is:

Performance Evaluation Standard:

T / C (%)> 60 is ineffective;

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

1. Inhibitory effect on glioma of the human brain U251

As a positive control, carmustine, 40 mg / kg (Renmin Pharmaceuticals, Amino Acid Inc. Tianjin, P.R. China), administered by intraperitoneal injection, was used once. CPT was administered to four groups at doses of 0.6 mg / kg, 1.7 mg / kg, 5.0 mg / kg, 15.0 mg / kg, respectively, by intraperitoneal injection. This treatment included ten injections.

The experimental results are shown in tables 1 and 2 and in figures 1 and 2. They show that CPT has a significant inhibitory effect on the growth of transplanted tumors (human brain glioma U251) in nude mice. Tumor inhibition intensities calculated on weight for the four treatment groups were 30.5%, 44.5%, 64.8%, and 87.5%, respectively. Compared to the control group, the mass of tumors in these treatment groups showed a significant or very significant statistical difference. The antitumor effect of CPT was significantly dose-dependent. The values of the relative intensity of the growth of T / C tumors (%) for the groups receiving 5.0 mg / kg and 15 mg / kg were <60. The relative tumor volume (RTV) for the group receiving 15 mg / kg was significantly different in statistics compared to this value in the control group.

table 2
The inhibitory effect of CPT on the growth of human glioma U251 (x ± SD) Group Number of animals Body weight (g) Tumor mass (g) Inhibition Rate (%) Start the end Start the end Negative control 8 8 21.9 ± 1.1 22.3 ± 2.6 1.28 ± 0.50 Karmustin
(the control) 8 7 22.8 ± 0.7 19.4 ± 2.1 0.09 ± 0.06 ^** 93.0 CPT 0.6 mg / kg 8 8 21.0 ± 0.8 21.0 ± 1.4 0.89 ± 0.49 30.5 CPT 1.7 mg / kg 8 8 21.6 ± 1.2 21.5 ± 2.0 0.71 ± 0.22 ^* 44.5 CPT 5.0 mg / kg 8 8 22.0 ± 1.1 21.9 ± 3.2 0.45 ± 0.26 ^** 64.8 CPT 15 mg / kg 8 8 21.6 ± 1.1 21.1 ± 1.7 0.16 ± 0.15 ^** 87.5

Table 3
The inhibitory effect of CPT on the growth of human glioma U251 (x ± SD) Group Tumor volume ^(mm3) RTV T / S (%) Start the end Negative control 165 ± 72 1238 ± 244 3.34 ± 2.28 Karmustin (control) 153 ± 35 109 ± 78 ^** 0.54 ± 0.29 ^** 16,0 CPT 0.6 mg / kg 141 ± 40 961 ± 582 2.77 ± 1.97 82.9 CPT 1.7 mg / kg 131 ± 34 588 ± 215 ^** 2.12 ± 1.29 63.5 CPT 5.0 mg / kg 133 ± 57 510 ± 333 ^** 1.32 ± 1.24 ^* 39.5 CPT 15 mg / kg 148 ± 58 261 ± 238 ^** 0.71 ± 0.57 ^** 21.3 * P <0.05 ** P <0.01 compared with the negative control group

2. Inhibitory effect on the growth of human lung cancer cells NCI-H460

As a positive control, cyclophosphamide (Shanghai Hualian Pharma Inc., P. R. China) was used, administered by intraperitoneal injection at a dose of 100 mg / kg. One additional injection was administered at a dose of 80 mg / kg after two weeks. CPT was administered to three treatment groups at doses of 1.7 mg / kg, 5.0 mg / kg, 15 mg / kg, respectively, by intraperitoneal injection once a day. In total, this treatment included ten injections.

The experimental results are shown in tables 4 and 5 and in figures 3 and 5. They show that CPT has a significant inhibitory effect on the growth of transplanted tumors (human lung cancer NCI-H460) in nude mice. The tumor inhibition rate calculated for the weight for the three treatment groups was 52.2%, 74.5% and 87.0%, respectively. Compared with the control group, the masses of tumors in these treatment groups showed a significant or very significant statistical difference. The values of the relative intensity of tumor growth (%) for groups receiving 5.0 mg / kg and 15 mg / kg were <60; relative tumor volumes (RTV) were significantly different in statistics compared to this value in the control group.

Table 4
Inhibitory effect of CPT on human lung cancer cell growth NCI-H460 Group Number of animals Body weight (g) Tumor mass (g) Inhibition rate
(%) Start the end Start the end Negative control 8 7 21.4 ± 0.9 19.3 ± 1.0 1.61 ± 0.65 Cyclophosphamide
(the control) 8 7 20.0 ± 1.3 16.1 ± 2.2 0.96 ± 0.35 ^* 40,4 CPT 1.7 mg / kg 8 7 20.1 ± 1.9 19.8 ± 1.6 0.77 ± 0.19 ^** 52,2 CPT 5.0 mg / kg 8 8 20.4 ± 1.9 18.5 ± 2.5 0.41 ± 0.28 ^** 74.5 CPT 15 mg / kg 8 8 20.9 ± 1.1 21.1 ± 1.7 0.21 ± 0.10 ^** 87.0

Table 5
Inhibitory effect of CPT on human lung cancer cell growth NCI-H460 Group Tumor volume ^(mm3) RTV T / S (%) Start the end Negative control 169 ± 58 1874 ± 637 5.98 ± 4.05 Cyclophosphamide
(the control) 179 ± 52 1121 ± 434 3.67 ± 2.15 61,4 CPT 1.7 mg / kg 200 ± 103 706 ± 170 ^* 2.27 ± 1.13 ^* 38,0 CPT 5.0 mg / kg 187 ± 51 469 ± 359 ^** 1.49 ± 0.76 ^** 24.9 CPT 15 mg / kg 192 ± 63 189 ± 102 ^** 0.79 ± 0.25 ^** 13,2 * P <0.05 ^** P <0.01 compared with the negative control group

Histopathological test results are shown in FIG. 4-1 - FIG. 4-6. Figures 4-1, 4-3, and 4-5 were a negative control group. Figure 4-1 shows that the tumor tissue diffused and was similar to clusters. In the center of the tumor, a large diluted necrosis was observed. The tumor was surrounded by a thin membrane of connective tissue and was poorly infiltrated by lymphocytes. Figure 4-3 shows that the tumor cells were located in the form of cluster-like and stiff-like structures. Tumor tissue was enriched in capillaries and was not infiltrated by lymphocytes. Figure 4-5 shows that the tumor cells were round or polygonal, had large nuclei and had little cytoplasm. Chromatin was loose, the nuclei were transparent with 2-3 nucleoli, which showed numerous signs of mitosis. Figures 4-2, 4-4 and 4-6 belong to the group, which was administered 15 mg / kg CPT. Figure 4-2 shows that the tumor tissue was solid clusters and a plate-like necrosis was visible in the center. In this tumor, there was a large amount of proliferated connective tissue and a thin membrane of connective tissue was around the tumor. The tumor was poorly infiltrated by lymphocytes. Figure 4-4 shows that the tumor cells were located in the form of stiff-like structures. Tumor tissue contained few capillaries and had necrosis in the center. She was surrounded by a connective tissue sheath and was infiltrated with a small amount of lymphocytes. In FIG. Figures 4-6 show that the tumor cells were round or polygonal, the nuclei were large and contained little cytoplasm. Chromatin was loose, with signs of mitosis. The tumor contained few capillaries and had necrosis with multiple small foci in the form of dots.

It is clear that the drug of the present invention had a strong inhibitory effect on human lung cancer cells NCI-H460.

3. Inhibitory effect on the growth of COLO 205 cells of human colon cancer

An injectable hydroxycamptothecin (trade name: Xi Su, Wuhan Lishizhen Pharmaceuticals Inc., Huangshi Lishizhen Pharmaceuticals Group, P. R. China) was used as a positive control, administered intraperitoneally at a dose of 1 mg / kg once a day. Administration was completed after 15 days of consecutive administrations. Significant tumor growth was observed; thus, hydroxycamptothecin was administered intraperitoneally at a dose of 100 mg / kg for another week after 8 days. A single administration of 80 mg / kg of hydroxycamptothecin was additionally carried out after two weeks. CPT was administered to three treatment groups at doses of 1.7 mg / kg, 5.0 mg / kg, 15 mg / kg, respectively, by intraperitoneal injection once a day. In total, this treatment included 15 injections.

The experimental results are shown in tables 6 and 7 and in figures 6 and 7-1 to 7-5, where FIG. 7-1 belongs to the group of negative control; FIG. 7-2 refers to the group that was administered 15.0 mg / kg CPT; FIG. 7-3 refers to the group that was administered 5.0 mg / kg CPT; FIG. 7-4 refers to the group to which 1.7 mg / kg CPT was administered; FIG. 7-5 belongs to the positive control group with hydroxycamptothecin. The results showed that CPT showed a significant inhibitory effect on the growth of a transplanted tumor (COLO 205 human rectum cancer) in nude mice. Tumors were significantly reduced in size in the group that received high doses of CPT after five days of administration. Some tumors even disappeared with an increase in the number of administrations. Observation of these mice was carried out for four weeks after cessation of administration and no tumor was observed in 1 of 3 animals. Macroscopic anatomical analysis of mice found only some signs of inoculation under the skin. The tumor inhibition rates based on weight for the three treated groups were 79.6%, 90.8%, and 97.4%, respectively. Compared with the control group, the masses of tumors in these treatment groups showed a significant or very significant statistical difference. The values of the relative intensity of tumor growth T / C (%) for the treated groups were <60; relative tumor volumes (RTV) were significantly different in statistics compared to that in the control group. In particular, the T / C value (%) for the group treated with a high dose of CPT was <10, which indicated that CPT was highly active against this type of tumor.

Table 6
The inhibitory effect of CPT on the growth of human colon cancer cells COLO 205 Group Number of animals Body weight (g) Tumor mass (g) Inhibition rate
(%) Start the end Start the end Negative control 8 8 19.5 ± 0.8 21.9 ± 1.9 1.96 ± 0.73 Hydroxicam ptothecin
(the control) 8 8 20.4 ± 0.9 21.0 ± 2.5 0.66 ± 0.26 ^** 66.3 CPT 1.7 mg / kg 8 8 20.6 ± 1.1 20.6 ± 3.0 0.40 ± 0.33 ^** 79.6 CPT 5.0 mg / kg 8 7 20.2 ± 0.8 21.4 ± 1.2 0.18 ± 0.15 ^** 90.9 CPT 15 mg / kg 8 8 20.0 ± 0.5 21.1 ± 2.4 0.05 ± 0.05 ^** 97.4

Table 7
The inhibitory effect of CPT on the growth of human colon cancer cells COLO 205 Group Tumor volume ^(mm3) RTV T / S (%) Start the end Negative control 151 ± 79 2532 ± 1190 8.30 ± 5.53 Hydroxysam-ptothecin
(the control) 150 ± 34 845 ± 66 ^** 3.92 ± 1.91 ^* 47.2 CPT 1.7 mg / kg 205 ± 47 582 ± 408 ^** 1.62 ± 0.62 ^** 19.5 CPT 5.0 mg / kg 180 ± 63 314 ± 175 ^** 0.91 ± 0.35 ^** 11.0 CPT 15 mg / kg 192 ± 81 52 ± 53 ^** 0.33 ± 0.27 ^** 4.0 * P <0.05 ^** P <0.01 compared with the negative control group

Industrial applicability

Tetrazolium recovery analysis (MTT) was used to determine the anticancer effect of the drug. Dose response curves were obtained by plotting the cell inhibition intensities and various drug concentrations and the inhibition concentrations of 50% of the cells (IC ₅₀ ) were calculated. Observations on numerous human tumor cell lines have shown that the composition of CPT has a significant inhibitory effect on tumor cell growth. CPT showed very significant in vitro cytolysis activity on the cells of more than 10 tumors, including human lung cancer, colon cancer, breast cancer, stomach cancer, pancreatic cancer, glioma, blood cell cancer, bladder cancer, prostate cancer colorectal colon cancer, cervical cancer and brain tumor cells. Inhibition concentrations of 50% cells (IC ₅₀ ) CPT for U251 (human brain glioma cells), COLO 205 (human colon cancer cells), NCI-H460 (human lung cancer cells), MDA-MB-435 (breast cancer cells human) were <0.01 μg / ml. IC ₅₀ CPT values for 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 <0, 1 mcg / ml. IC ₅₀ CPT for RPMI 8226 (human multiple myeloma cells) is <1 μg / ml. CPT also exhibits some inhibitory effect on the growth of SCLC (human small cell lung cancer cells).

In vivo experimental results show that intraperitoneal administration of CPT at a dose of 15 mg / kg / day for 10-15 consecutive days showed a very significant inhibitory effect on the growth of tumor cells, including the growth of human colon cancer COLO 205, human lung cancer NCI-H460, human gliomas U251, etc.

Compared to the negative control group, which did not receive the drug, CPT can significantly slow down the growth of tumors in animals, and a high dose at the early stage of growth of the transplanted tumor can lead to a decrease in the volume of the tumor or even to its disappearance. The effect of CPT is clearly dose dependent. In addition, the effectiveness is to some extent related to the course of treatment. In models of nude mice that received transplantation of human colorectal tumor cells COLO 205, it was observed that the tumor was smaller than the size before administration, even four weeks after cessation of administration, in which these mice received 15 consecutive injections of CPT at a dose of 15 mg / kg / day. The presence of tumors could not be confirmed at the inoculation sites in 1 of 3 animals, and only signs caused by inoculation were observed during anatomical analysis.

All these data indicate that the administration of CPT showed a significant inhibitory effect on the growth of transplanted human tumors in nude mice. Compared with the control group for U251, the inhibition rate of 15 mg / kg CPT based on the tumor mass was 87.5% and 87.7%, respectively; for NCI-H460, the inhibition rate was 87.0% and 88.8%, respectively; for COLO 205, the inhibition rate was 97.4% and 97.8%, respectively, indicating a very significant statistical difference. The invention used three models for the experiment. The results showed that relative tumor volumes (RTV) in these three animal models showed a statistically significant difference compared with the negative control group, where CPT was continuously administered to these animals at a dose of 5.0 mg / kg for 10-15 days. The data for the group treated with 15 mg / kg CPT significantly differed from the data of the negative control group, with all T / C values (%) being less than 25, sometimes even less than 10, which indicates that CPT was high antitumor activity, and these experiments had good reproducibility.

CPT did not show any side or toxic effects in animals. No significant difference was observed in the parameters related to the life status of mice, such as body weight and viability of the treated groups compared to the same parameters of the negative control group, except that the size of the tumor in the treated mice was smaller. Based on the above, it was demonstrated that CPT has a significant selective inhibitory effect on tumor cells and that CPT does not induce apoptosis of normal tissue cells. Thus, CPT can be used as an effective and safe drug for the treatment of cancer. CPT is both theoretically and industrially important and has a promising future in the market.

Claims (14)

1. Recombinant protein having an anti-cancer effect, which is selected from the group consisting of: 1) a protein with the amino acid sequence shown in SEQ ID NO: 2; 2) a protein whose amino acid sequence is more than 90% homologous to the amino acid sequence shown in SEQ ID NO: 2, and which has the same activity as the protein with the sequence shown in SEQ ID NO: 2; 3) a protein obtained by substitution, deletion or addition of one or more amino acid residues to the amino acid sequence shown in SEQ ID NO: 2, and which has the same activity as the protein with the sequence represented by bSEQ ID NO: 2. 2. The protein according to claim 1, characterized in that the protein has the amino acid sequence shown in SEQ ID NO: 2. 3. The gene encoding the recombinant protein according to claim 1, selected from the group consisting of: 1) DNA with the nucleotide sequence shown in SEQ ID NO: 1; 2) DNA encoding a protein with the amino acid sequence shown in SEQ ID NO: 2; 3) DNA with a nucleotide sequence that is more than 90% homologous to the DNA sequence shown in SEQ ID NO: 1, and which encodes a protein with the same activity as the protein encoded by DNA with the sequence represented in SEQ ID NO: 1 ; 4) DNA encoding a protein obtained by substitution, deletion or addition of one or more amino acid residues to the amino acid sequence shown in SEQ ID NO: 2, and which has the same activity as the protein with the sequence shown in SEQ ID NO: 2 . 4. The gene according to claim 3, characterized in that its nucleotide sequence is presented in SEQ ID NO: 1. 5. A medicine for the treatment of cancer, containing as an active ingredient the recombinant protein according to claim 1 or 2. 6. The expression vector containing the gene according to claim 3 or 4. 7. The cell line containing the gene according to claim 3 or 4, designed for expression of a recombinant protein. 8. The use of the recombinant protein according to claim 1 or 2 for the manufacture of a medicament for the treatment of cancer. 9. The use of claim 8, wherein said cancer is selected from the group consisting of colorectal cancer, lung cancer, multiple myeloma, brain glioma, leukemia, breast cancer, small cell lung cancer, and pancreatic cancer. 10. The use of claim 9, wherein said cancer is multiple myeloma. 11. The use of claim 9, wherein said cancer is leukemia. 12. The use of claim 9, wherein said cancer is a glioma of the brain. 13. The use of claim 9, wherein said cancer is lung cancer. 14. The use of claim 9, wherein said cancer is rectal cancer.

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