WO2010037284A1

WO2010037284A1 - Polypeptides for treating or preventing cancers, the derivative products and uses thereof

Info

Publication number: WO2010037284A1
Application number: PCT/CN2009/073201
Authority: WO
Inventors: 陈建华; 黄毅; 熊骏宇; 陈彩虹
Original assignee: 武汉凯泰新生物技术有限公司
Priority date: 2008-09-26
Filing date: 2009-08-12
Publication date: 2010-04-08

Abstract

Provided are polypeptides with the amino acid sequences shown as SEQ ID NO:1 and SEQ ID NO:2, pharmaceutical compositions comprising said polypeptides, genes encoding said polypeptides, expression vectors and host cells comprising said genes, as well as uses of said polypeptides for manufacturing medicaments for the treatment of cancers. Said polypeptides can be used alone to treat or prevent cancers and can enhance markedly the ability of chemotherapeutics to kill cancer cells selectively when used combined with chemotherapeutics, with little cytotoxicity to normal cells, thus effectively decrease the toxicity and side-effect of chemotherapeutics. Also provided are products of said polypeptides conjugated with agent which can enhance the accumulation of said polypeptides in cells.

Description

Polypeptides for treating or preventing cancer, and derivatives thereof and applications thereof

Technical field

The present invention relates to polypeptides and uses thereof, and more particularly to polypeptides capable of treating or preventing cancer, and to a product having therapeutic or preventive effects derived from the peptides, and to the field of biomedicine. Background technique

In recent years, the incidence of malignant tumors in China has shown a clear upward trend, and tumors have become one of the major diseases that seriously threaten human safety and health. According to the statistics of the Ministry of Health of China, the total number of new cancer patients in China is about 2.2 million per year. Among them, there are about 1.21 million new cases of malignant tumors per year, accounting for 55% of the total number of new cancer patients in China every year. There are about 3.1 million existing tumor patients, of which about 1.82 million are malignant tumors, accounting for 58% of the total number of existing cancer patients in China. At present, chemotherapy is still one of the main means of cancer treatment. It is foreseeable that as the incidence of cancer increases, the demand for anti-cancer drugs will further increase.

Cisplatin is the first metal complex discovered in the 1960s and has antitumor activity. In 1971, cisplatin was clinically proven to have a strong inhibitory effect on certain tumors in humans. Cisplatin has a broad anti-tumor spectrum and is currently the first-line drug for the treatment of breast cancer, ovarian cancer, and testicular cancer. Li Yixin, "The Drug of Choice for Cancer Chemotherapy", Foreign Medicine-Synthetic Drug Biochemical Drugs, 89- 101, 19, 1998), and often combined with other chemotherapeutic drugs such as etoposide, bleomycin, doxorubicin and 5-fluorouracil for the treatment of head and neck cancer and stomach cancer. The main disadvantages of cisplatin anticancer drugs are: (1) severe side effects: including nephrotoxicity, gastrointestinal toxicity, ototoxicity and neurotoxicity; (2) low activity on certain tumor cells: such as breast cancer , colon cancer, etc.; (3) easy to produce drug resistance; (4) small water solubility, not easy to metabolize in the body; (5) must be injected (Yu Bangliang et al, "platinum anticancer drug research progress" Hainan University Journal Nature Scientific edition, 72-80, 23, 2005). Therefore, it is a research goal of the medical profession to improve the efficacy of anti-tumor chemotherapy drugs and reduce the toxicity of drugs to normal tissue cells.

It is known from the Chinese patent application published under the number CN 1816564A (publication date: August 9, 2006) that RasGAP ₃₁₇ _ ₃₂₆ peptide can enhance the ability of cisplatin to kill cancer cells, and RasGAP ₃₁₇ _ ₃₂₆ peptide must and can kill cancer. The combination of the chemotherapeutic drugs of the cells can function, and the sensitization of the peptide is limited; and when the concentration of cisplatin is Ο μ 它, it has no inhibitory effect on cancer cells. Summary of the invention

The object of the present invention is to overcome the deficiencies of the prior art and to provide a polypeptide capable of treating or preventing cancer and a derivative thereof, which can be used not only for treating or preventing cancer, but also when combined with a genetic toxin. It can significantly enhance the ability of genetic toxins to selectively kill cancer cells.

The inventors have found through intensive studies that a polypeptide having the characteristics listed in the following technical means can achieve the above object.

A polypeptide capable of treating or preventing cancer, the amino acid sequence of which is represented by SEQ ID NO.

A polypeptide capable of treating or preventing cancer, the amino acid sequence of which is represented by SEQ ID N0.2.

An isolated and purified nucleotide sequence encoding a polypeptide of the amino acid sequence set forth in SEQ ID NO. 1 or SEQ ID NO.

An expression vector comprising at least one copy of the nucleotide sequence encoding the polypeptide of SEQ ID NO. 1 or SEQ ID N0.2.

A prokaryotic or eukaryotic host cell comprising the above expression vector.

The present invention still further provides a product obtained by conjugating or mixing the polypeptide of SEQ ID NO. 1 or SEQ ID N0.2 with a preparation capable of increasing its accumulation in cells.

The above-described formulation conjugated to the peptide is a carrier which assists the peptide in penetrating the cell membrane. The peptide of SEQ ID NO. 1 or SEQ ID N0.2 and a carrier capable of assisting the peptide to penetrate the cell membrane, such as HIV48-57 peptide, FHV-exo 35-49 peptide, HTLV-II Rex 4-16 peptide or BMV The gag7-25 peptide is conjugated, and the compound produced by the conjugation can effectively pass through the cell membrane and act locally in the cancer cells, thereby having a stronger effect of killing cancer cells.

The preparation conjugated to the polypeptide of SEQ ID NO. 1 or SEQ ID N0.2 may also be a nanomaterial, a liposome or an oily compound.

The polypeptide of SEQ ID NO. 1 or SEQ ID N0.2 is conjugated to a high molecular material such as a nanomaterial or a liposome, and the compound formed by the conjugation can cause the polypeptide of the present invention to be transported more stably in the body. Go to target cells. The polypeptide of the present invention may also be mixed with an oily compound or a mixture of a plurality of oily compounds, and the resulting mixture may also allow the polypeptide of the present invention to be more stably transported to the target cells in the body.

A pharmaceutical composition for treating or preventing cancer, comprising: a pharmaceutically effective amount as an active substance The peptide of SEQ ID NO. 1 or SEQ ID N0.2 is comprised of a pharmaceutically acceptable carrier, diluent and/or adjuvant.

The invention further provides the use of a polypeptide of SEQ ID NO. 1 or SEQ ID N0.2 for the manufacture of a medicament for the treatment or prevention of cancer.

The cancer includes, but is not limited to, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, esophageal cancer, breast cancer, leukemia, bladder cancer, cervical cancer or nasopharyngeal cancer.

The present invention also provides the use of the polypeptide of SEQ ID NO. 1 or SEQ ID N0.2 for the preparation of a medicament for enhancing the killing of cancer cells by a genetic toxin.

The cancer cells include, but are not limited to, cancer cells such as lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, esophageal cancer, breast cancer, leukemia, bladder cancer, cervical cancer or nasopharyngeal cancer.

The genetic toxins include, but are not limited to, cisplatin, oxaliplatin, paclitaxel, epirubicin, doxorubicin, pirarubicin, daunorubicin, mitomycin, dacarbazine, Cyclophosphamide, gemcitabine or capecitabine.

The polypeptide of the present invention can be mixed with a genetic toxin and then added with a pharmaceutically acceptable adjuvant or carrier to obtain a more effective anticancer drug.

The polypeptides of the invention are typically employed in amounts which achieve the intended purpose. When used for the prevention and treatment of cancer, the polypeptide of the present invention or a pharmaceutical composition thereof is administered or used in a therapeutically effective amount. For systemic administration, a therapeutically effective amount or amount can be estimated based on in vitro experiments, and the starting dose in vivo of the polypeptide of the present invention can also be estimated by animal models using methods commonly employed in the art. Of course, the particular effective amount of the polypeptide of the present invention will depend on the particular subject being treated, the weight of the subject being treated, the severity of the disease, the mode of administration, and the clinical judgment of the attending physician.

The inventors have found through intensive studies that the above polypeptides are useful for treating or preventing cancer. The polypeptide not only has obvious lethal effect on cancer cells when used alone, but also can be combined with clinically used chemotherapy drugs (such as cisplatin) to significantly increase the sensitivity of chemotherapy drugs to cancer cells and enhance their cancer resistance. The lethality of the cell reduces its dosage. The polypeptide of the invention can kill various cancer cells such as lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, esophageal cancer, breast cancer and leukemia. The polypeptide of the present invention has an anti-tumor effect; the polypeptide of the present invention and cisplatin have a stronger effect on cancer cells than RasGAP _317. ₃₂₆ peptide (RasGAP _317. ₃₂₆ peptide amino acid residue sequence: WMWVTNLRTD) and cis-chlorine The effect of combining ammonia and platinum. The polypeptide of the invention has no obvious toxicity enhancing effect on normal cells, and the invention The polypeptide involved can be prepared by chemical synthesis, has high purity, small molecular weight, strong specificity, no immunogenicity, and is safe and reliable.

The polypeptide of the present invention can be used alone for the treatment and prevention of cancer and has good effects. The polypeptide of the present invention can also be combined with a chemotherapeutic drug (such as cisplatin, paclitaxel, etc.), and can selectively enhance the sensitivity of the chemotherapeutic drug to tumor cells. , significantly reducing the onset dose of chemotherapy drugs, due to the small toxic effect on normal cells, thereby reducing the toxicity and side effects of chemotherapy drugs. DRAWINGS

1 shows purified by HPLC after RasGAP ₃₁₇ - ₃₂₆ of FIG characteristic peaks analytical column eluted peptide. Figure 2 is a graph showing the characteristic peaks eluted on the analytical column of the polypeptide of SEQ ID NO. 1 after HPLC detection and purification.

Figure 3 is a graph showing the characteristic peaks eluted on the analytical column of the polypeptide of SEQ ID NO. 2 after HPLC detection and purification.

₃₂₆ peptide and cisplatin are the results of inhibition of tumor cells - 1, SEQ ID NO 2, RasGAP 317 of FIG. 4 SEQ ID NO..

After ₃₂₆ peptide cisplatin were combined inhibitory effect on tumor cells results - 1, SEQ ID NO 2, RasGAP 317 in FIG. 5 SEQ ID NO..

Figure 6 SEQ ID NO 1, SEQ ID NO 2, RasGAP 317 -. 326 peptide cisplatin inhibition results after normal cells were combined.

₃₂₆ peptide and cisplatin inhibition results are normal cells - 1, SEQ ID NO 2, RasGAP 317 in FIG. 7 SEQ ID NO..

Figure 8 shows the results of inhibition of gastric cancer cells by the combination of the peptide of SEQ ID NO. 1 and cisplatin.

Figure 9 Results of inhibition of lung cancer cells by the combination of the SEQ ID NO. 1 peptide and cisplatin.

Figure 10 Results of inhibition of colon cancer cells by the combination of the SEQ ID NO. 1 peptide and cisplatin. Figure 11 Results of inhibition of colon cancer cells by the combination of the SEQ ID NO. 2 peptide and cisplatin.

FLKGDMFIVHNELEDG FLKGDMFIVHNELEDGWMWVT detailed description

The invention will be further described in conjunction with the specific embodiments, and the advantages and features of the invention will become more apparent. However, these examples are merely exemplary and do not constitute any limitation on the scope of the invention. It should be understood by those skilled in the art that the details and the details of the present invention may be modified or substituted without departing from the spirit and scope of the invention. Example 1 RasGAP _317. Synthesis of ₃₂₆ peptide

1) Experimental instruments and materials:

Dimethylformamide (DMF), piperidine, resin, dichloromethane (DCM), Kaiser kit, 1-hydroxybenzotriazole (HOBt), tetramethylurea hexafluorophosphate (HBTU), dichotomous Propylethylamine (DIEA), methanol, various amino acids, peptide solid phase synthesis tubes.

2) Experimental steps:

The resin was weighed and put into a peptide solid phase synthesis tube (hereinafter referred to as a reactor), and an appropriate amount of DMF was added to swell for 10 minutes. The DMF was removed to carry out the Fmoc deprotection reaction, that is, an appropriate amount of a 20% piperidine-containing DMF solution was added, and the mixture was thoroughly stirred. After 5 minutes, the solution was withdrawn and re-added to a 20% piperidine in DMF solution for 7 minutes. The protective solution was removed, the resin was washed 4-5 times with DMF, DMF was withdrawn, and washed with DCM 1-2 times. A small amount of resin (about 5-10 mg) was taken from the reactor and washed twice with ethanol. The Kaiser method detects and records the color, prepares the feed, and enters the amino acid condensation reaction. According to the amino acid sequence of RasGAP ₃₁₇ _ ₃₂₆ peptide, the corresponding amino acid and HOBt were taken in a suitable container, dissolved in DMF, dissolved completely, and then added to DIEA for 5 minutes. The weighed HBTU was added and dissolved in an ice water bath for 5 minutes. Into the reactor, the reaction was stirred. After 90 minutes, a small amount of resin was taken from the reactor in a test tube and washed twice with ethanol, and detected by Kaiser method. The liquid in the reactor was withdrawn, washed twice with DMF, and DMF was withdrawn to obtain a peptide resin after condensation of the first amino acid. The resulting peptide resin was repeatedly carried out "deprotection Fmoc - amino acid condensation" reaction step, the last amino acid to the completion of the reaction, to give RasGAP ₃₁₇ _ ₃₂₆ peptide. With the above method, in accordance with the sequence of HIV ₄₈ _ ₅₇ chemically synthesized peptide corresponding to the connection continues on the RasGAP amino acids ₃₁₇ _ ₃₂₆ peptide, obtained conjugated HIV RasGAP ₄₈ _ ₅₇ _ ₃₁₇ ₃₂₆ peptide. After the reaction was completed, DCM washed the resin 2-3 times, the solvent was removed, and the mixture was stirred by methanol (5 min + 5 min), the methanol was withdrawn, and the pumping was continued for 15-20 min. The synthesized peptide resin was taken out from the reactor, transferred to a round bottom flask, drained in a desiccator, and lysed at room temperature. Time. The resin was filtered and the solution was concentrated and lyophilized under vacuum. The crude peptide was purified by Shimadzu LC-6AD preparative reverse phase HPLC using a HPLC >90% purity. The pure peptide obtained was identified by mass spectrometry (MS, electrospray) and its molecular weight was 2813.29, which was consistent with the theoretically calculated value of 2813.29. Example 2 Synthesis of the peptide described in SEQ ID NO.

1) Experimental instruments and materials:

2) Experimental steps:

The resin was weighed and put into a peptide solid phase synthesis tube (hereinafter referred to as a reactor), and an appropriate amount of DMF was added to swell for 10 minutes. The DMF was removed to carry out the Fmoc deprotection reaction, that is, an appropriate amount of a 20% piperidine-containing DMF solution was added, and the mixture was thoroughly stirred. After 5 minutes, the solution was withdrawn and re-added to a 20% piperidine in DMF solution for 7 minutes. The protective solution was removed, the resin was washed 4-5 times with DMF, DMF was withdrawn, and washed with DCM 1-2 times. A small amount of resin (about 5-10 mg) was taken from the reactor and washed twice with ethanol. The Kaiser method detects and records the color, prepares the feed, and enters the amino acid condensation reaction. The corresponding amino acid, HOBt was weighed in the appropriate container according to the sequence of the amino acid sequence of the peptide described in SEQ ID NO. 1, dissolved in DMF, dissolved completely, added to DIEA for 5 minutes, added to the weighed HBTU, dissolved in ice water bath. It was activated for 5 minutes, put into the reactor, and stirred. After 90 minutes, a small amount of resin was taken from the reactor in a test tube and washed twice with ethanol, and detected by Kaiser method. The liquid in the reactor was withdrawn, washed twice with DMF, and DMF was withdrawn to obtain a peptide resin after condensation of the first amino acid. The above "Fmoc deprotection-amino acid condensation" reaction step is repeated on the obtained peptide resin, and the last amino acid sequence of the sequence of SEQ ID NO. 1 is completed to obtain the peptide of SEQ ID NO. With the above methods, the peptide according to the sequence of HIV in ₅₇ SEQ ID NO. 1 connected to the corresponding peptide synthesis continues chemical amino acid, to obtain conjugated HIV SEQ ID NO. 1 ₄₈ _ ₅₇ peptide. After the reaction was completed, DCM washed the resin 2-3 times, the solvent was removed, and methanol was added to agitation (5 min + 5 min), the methanol was withdrawn, and the extraction was continued for 15-20 min. The synthesized peptide resin was taken out from the reactor, transferred to a round bottom flask, drained in a desiccator, and lysed at room temperature for two hours. The resin was filtered and the solution was concentrated and lyophilized under vacuum. The crude peptide was purified by Shimadzu LC-6AD preparative reverse phase HPLC using a HPLC >90% purity. The obtained pure peptide was identified by mass spectrometry (MS, electrospray), and its molecular weight was 3411.01, and the theoretical calculation value. 3412.96 is consistent.

The FHV-exo 35^9 peptide, HTLV-II Rex _Φ16 peptide or BMV gag ₂₅ peptide and the SEQ ID NO. 1 peptide of the present invention can be conjugated as described above. Example 3 Synthesis of the peptide described in SEQ ID NO.

1) Instruments and materials:

2) Experimental steps:

The resin was weighed and put into a peptide solid phase synthesis tube (hereinafter referred to as a reactor), and an appropriate amount of DMF was added to swell for 10 minutes. The DMF was removed to carry out the Fmoc deprotection reaction, that is, an appropriate amount of a 20% piperidine-containing DMF solution was added, and the mixture was thoroughly stirred. After 5 minutes, the solution was withdrawn and re-added to a 20% piperidine in DMF solution for 7 minutes. The protective solution was removed, the resin was washed 4-5 times with DMF, DMF was withdrawn, and washed with DCM 1-2 times. A small amount of resin (about 5-10 mg) was taken from the reactor and washed twice with ethanol. The Kaiser method detects and records the color, prepares the feed, and enters the amino acid condensation reaction. The amino acid sequence of the peptide according to SEQ ID NO. 2 is weighed and the corresponding amino acid, HOBt is weighed in a suitable container, dissolved in DMF, dissolved completely, added to DIEA for 5 minutes, added to the weighed HBTU, dissolved in ice water bath to activate 5 In a minute, put it into the reactor and stir the reaction. After 90 minutes, a small amount of resin was taken from the reactor in a test tube and washed twice with ethanol, and detected by Kaiser method. The liquid in the reactor was withdrawn, washed twice with DMF, and DMF was withdrawn to obtain a peptide resin after condensation of the first amino acid. The above "Fmoc deprotection-mono-amino acid condensation" reaction step is repeated on the obtained peptide resin until the last amino acid sequence of the sequence of SEQ ID NO. 2 is completed, and the peptide of SEQ ID NO. 2 is obtained. With the above method, in accordance with the sequence of peptide synthesis continues HIV ₅₇ connected to the corresponding amino acid chemistry, to obtain conjugated HIV ₄₈ _ ₅₇ of SEQ ID NO. 2 peptide in SEQ ID NO. 2 peptide. After the reaction was completed, DCM washed the resin 2-3 times, the solvent was removed, and methanol was added to agitation (5 min + 5 min), the methanol was withdrawn, and the extraction was continued for 15-20 min. The synthesized peptide resin was taken out from the reactor, transferred to a round bottom flask, drained in a desiccator, and lysed at room temperature for two hours. The resin was filtered and the solution was concentrated and lyophilized under vacuum. The crude peptide was purified by Shimadzu LC-6AD preparative reverse phase HPLC using a HPLC >90% purity. The obtained pure peptide was identified by mass spectrometry (MS, electrospray) and its molecular weight was 4660.41, which was in agreement with the theoretical calculation. The FHV-exo 35^9 peptide, HTLV-II Rex _Φ16 peptide or BMV gag ₂₅ peptide and the SEQ ID NO. 2 peptide of the present invention can be conjugated as described above. Experimental Example 1 MTT assay for SEQ ID NO. 1, SEQ ID N0.2, and RasGAP _317. Inhibition of tumor cells by ₃₂₆ peptides

I. Experimental sample: The polypeptide synthesized in Examples 1-3: RasGAP _317. ₃₂₆ peptide, SEQ ID NO. 1 peptide, SEQ ID NO. 2 peptide;

Second, the experimental method

The human cervical cancer cell line Hela (purchased from the China Center for Type Culture Collection, Wuhan University Center) was suspended in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum, and seeded at 96-well at a density of 5000-10000 cells/well. board. The cells were cultured at 37 ° C, 5% CO ₂ . When the cell density reaches 70%-80%, cisplatin, RasGAP _317. ₃₂₆ peptide, peptide of SEQ ID NO. 1, or peptide of SEQ ID NO. 2, wherein the concentration gradient of each drug is 40μιηο1/ί, 30μιηο1/ί, 20μιηο1/ί, μμηιοΐ/L, 3 duplicate wells per concentration. After 20 hours of incubation, MTT (5 mg/ml) 20 μl was added to each well. After 4 hours, the culture solution was aspirated, and dimethyl sulfoxide (DMSO) 150 μl was added to each well, and the purple crystals were sufficiently dissolved by shaking for 10 minutes. The absorbance was measured at 570 nm by a microplate reader.

The cell inhibition rate is calculated as follows:

Cell inhibition rate = (empty control group OD value - experimental group OD value) / blank control group OD value

X 100%

Data statistics are expressed as mean soil standard deviation using the t test.

Third, the experimental results:

As can be seen from Figure 4, the inhibition of Hela cells by the polypeptides of SEQ ID N0.1 and SEQ ID N0.2 was significantly greater than that of the RasGAP ₃₁₇ ₃₂₆ peptide. When the drug is at a low concentration, ie, the concentration is less than ΙΟμιηοΙ/L, the peptides of SEQ ID NO.1 and SEQ ID NO.2 have an inhibitory effect on Hela cells and are more than the same concentration of cisplatin or RasGAP ₃₁₇ _ The effect of the ₃₂₆ peptide was more pronounced, and the difference between the two was significant and statistically significant. The peptides of SEQ ID N0.1 and SEQ ID NO. 2 have significant inhibitory effects on HeLa cells, while the same concentration of RasGAP ₃₁₇ _ ₃₂₆ peptide has little inhibitory effect on HeLa cells. The polypeptide of the present invention can be used as a drug for treating and preventing cancer. Experimental Example 2 MTT assay for the effect of different peptides and cisplatin on tumor cells

Second, the experimental method

The human cervical cancer cell line Hela was suspended in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum, and seeded in a 96-well plate at a density of 5000-10000 cells/well. After 24 hours of culture, a mixture of cisplatin and RasGAP ₃₁₇ _ ₃₂₆ peptide, cisplatin and a peptide mixture of SEQ ID NO. 1, or cisplatin and the peptide of SEQ ID NO. 2 were added, respectively. Mixture. The concentration of cisplatin in each mixture was 1.1μιηο1/ί, 0.37μηιο1/ί 0.12μιηο1/ί, Ομηιοΐ/L, and the final concentration of each peptide was 20 mol/L. . After 20 hours of incubation, MTT (5 mg/ml) 20 μl was added to each well. After 4 hours, the culture solution was aspirated, and DMSO 150 μl was added to each well, and the purple crystals were sufficiently dissolved by shaking for 10 minutes. The absorbance was measured at 570 nm by a microplate reader.

The cell inhibition rate is calculated as follows:

X 100%

Data statistics are expressed as mean soil standard deviation using the t test.

Third, the experimental results:

As can be seen from Figure 5, when cisplatin is used in combination with SEQ ID N0.1 or in combination with SEQ ID NO. 2 or in combination with RasGAP ₃₁₇ _ ₃₂₆ peptide, their inhibitory effects on Hela cells are significantly stronger. By using cisplatin alone, it is indicated that the polypeptide of the present invention can increase the sensitivity of cisplatin to Hela cells and enhance its inhibitory effect on HeLa cells. The ability of the three peptides to increase the sensitivity of cisplatin to Hela cells is different in this experiment, wherein the peptide of SEQ ID NO. 2 has the most sensitizing effect on cisplatin, followed by SEQ ID NO. 1; the peptide; when the concentration of cisplatin is Ομηηοΐ / L, the peptide of SEQ ID NO. 1 and the peptide of SEQ ID NO. 2 inhibited HeLa cells by 17.6% and 17.7%, respectively. The inhibition rate of RasGAP ₃₁₇ . ₃₂₆ peptide is only 2.5%, and the peptide of SEQ ID NO. 1 and the peptide of SEQ ID NO. 2 are more sensitive to cisplatin than RasGAP ₃₁₇ _ ₃₂₆ peptide to cisplatin The sensitization of platinum is statistically significant. Experimental Example 3 MTT assay for inhibition of normal cells by different peptides and cisplatin I. Experimental sample: The polypeptide synthesized in Examples 1-3: RasGAP _317. ₃₂₆ peptide, SEQ ID NO. 1 peptide, SEQ ID NO. 2 peptide;

Second, the experimental method

Human myoblasts (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were suspended in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, and seeded in 96-well plates at a density of 5000-10000 cells/well. After 24 hours of culture, when the cell density reached 70%-80%, a mixture of cisplatin and RasGAP ₃₁₇ _ ₃₂₆ peptide, cisplatin and a mixture of peptides of SEQ ID NO. 1 were added, respectively. A mixture of cisplatin and the peptide of SEQ ID NO. The concentration gradient of cisplatin is 250μιηο1/ί, 200μιηο1/ί, 150μιηο1/ί, 100μιηο1/ί, 50 mol/L, Ομηιοΐ/L, and the final concentration of the polypeptide is 20 mol/L, and each concentration is set to 3 Multiple holes. After 20 hours of incubation, MTT (5 mg/ml) 20 μl was added to each well. After 4 hours, the culture solution was aspirated, and DMSO 150 μl was added to each well, and the purple crystals were sufficiently dissolved by shaking for 10 minutes. The absorbance was measured at 570 nm by a microplate reader.

The cell inhibition rate is calculated as follows:

X 100%

Third, the experimental results:

As can be seen from Fig. 6, when 50 μιηοοίί cisplatin and SEQ ID NO. 1 were used together or in combination with the peptide of SEQ ID N0.2, the inhibition rate to human myoblasts was 17% and 9.6%, using the same concentration of cisplatin alone, or the same concentration of cisplatin and RasGAP ₃₁₇ _ ₃₂₆ peptide, the inhibition rate was 25.3% and 29.1%. As the concentration of cisplatin is gradually increased, the inhibition of human myoblasts by cisplatin combined with SEQ ID NO. 1 or in combination with the peptide of SEQ ID NO is gradually enhanced, but is the same as used alone. There was no significant difference between the concentrations of cisplatin or the combination of the same concentrations of cisplatin and RasGAP ₃₁₇ _ ₃₂₆ peptides. The experimental results show that the polypeptide of the present invention and cisplatin have no toxic enhancement effect on normal cells, that is, the polypeptide of the present invention is non-toxic or less toxic to normal cells. Experimental Example 4 MTT assay SEQ ID NO 1, SEQ ID NO 2 and a RasGAP ₃₁₇ -. ₃₂₆ peptide inhibition experiments were normal cells.

I. Experimental sample: The polypeptide synthesized in Examples 1-3: RasGAP _317. ₃₂₆ peptide, SEQ ID NO. 1 peptide, SEQ ID NO. 2 peptide; Second, the experimental method

Human myoblasts (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were suspended in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, and seeded in 96-well plates at a density of 5000-10000 cells/well. After 24 hours of culture, when the cell density reaches 70%-80%, cisplatin, RasGAP _317. ₃₂₆ peptide, peptide of SEQ ID NO. 1, and peptide of SEQ ID NO. 2, each of which is added, respectively, are added. The concentration gradient of the drug was 80 μιηο1/ί, 40 μιηο1/ί, 20 μιηο1/ί, Ομηιοΐ/L, and 3 complex holes were set for each concentration. After 20 hours of incubation, MTT (5 mg/ml) 20 μl was added to each well. After 4 hours, the culture solution was aspirated, and DMSO 150 μl was added to each well, and the purple crystals were sufficiently dissolved by shaking for 10 minutes. The absorbance was measured at 570 nm by a microplate reader.

The cell inhibition rate is calculated as follows:

X 100% III. Experimental results

It can be seen from Fig. 7 that cisplatin has a significant killing effect on human myoblasts, while the same concentration of the peptide of SEQ ID NO. 1, the peptide of SEQ ID NO. 2, and the RasGAP ₃₁ w ₂₆ peptide pair The killing effect of myoblasts is small, that is, the peptide of SEQ ID NO. 1 of the present invention and the peptide of SEQ ID NO. 2 have no obvious killing effect on normal cells. Experimental Example 5 MTT assay for the effect of SEQ ID NO. 1 peptide and cisplatin on human gastric cancer SGC-7901 cells

1. Experimental sample: Example 2 synthesized SEQ ID NO. 1 peptide;

Second, the experimental method

The human gastric cancer cell line SGC-7901 (purchased from the China Center for Type Culture Collection, Wuhan University Center) was suspended in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum, and seeded at a density of 5000-10000 cells/well. Orifice plate. After 24 hours of culture, the control group was added with cisplatin, and the experimental group was added with cisplatin and the peptide mixture of SEQ ID NO. The concentration of cisplatin in the control group and the experimental group was 90μηιο1/ί 30μηιο1/ί ΙΟμηιοΙ/L, 3.3μηιο1/ί, l. ol/L, 0.37μηιο1/ί, SEQ ID NO. 1 in the experimental mixture. The final concentration of the peptide was 20 μιηο1/ί, and 3 replicate wells were set for each concentration. After 20 hours of incubation, MTT (5mg/ml) 20μ1 was added to each well. After 4 hours, the culture solution was aspirated and added per well. Into DMSO 150μ1, shake for 10 minutes to dissolve the purple crystals. The absorbance was measured at 570 nm by a microplate reader.

The cell inhibition rate is calculated as follows:

Cell inhibition rate = (blank control group OD value - experimental group 0D value) / blank control group 0D value X 100%

Third, the experimental results:

As can be seen from Figure 8, when cisplatin is used in combination with SEQ ID N0.1, their inhibitory effect on SGC-7901 cells is significantly stronger than that of cisplatin alone, indicating that SEQ ID N0.1 can increase cis-chlorine The sensitivity of ammonia platinum to SGC-7901 cells enhances its inhibitory effect on SGC-7901 cells. Experimental Example 6 MTT assay for the effect of SEQ ID NO. 1 peptide and cisplatin on human lung cancer A549 cells

1. Experimental sample: Example 2 synthesized SEQ ID NO. 1 peptide;

Second, the experimental method

The human lung cancer cell line A549 (purchased from the China Center for Type Culture Collection, Wuhan University Center) was suspended in F-12K medium containing 10% heat-inactivated fetal bovine serum, and seeded at a density of 5000-10000 cells/well. Orifice plate. After 24 hours of culture, the control group was added with cisplatin, and the experimental group was added with cisplatin and the peptide mixture of SEQ ID NO. The concentration of cisplatin in the control group and the experimental group was 90 μιη _Ο 1/ί, 30 μιηο1/ί, ΙΟμηιοΙ/L, 3.3 μηιο1/ί l. l mol/L, 0.37μηιο1/ί, SEQ in the experimental group. The final concentration of the peptides of ID NO. 1 was 20 μιηο1/ί, and three replicate wells were set for each concentration. After 20 hours of incubation, MTT (5 mg/ml) 20 μl was added to each well. After 4 hours, the culture solution was aspirated, and DMSO 150 μl was added to each well, and the purple crystals were sufficiently dissolved by shaking for 10 minutes. The absorbance was measured at 570 nm by a microplate reader.

The cell inhibition rate is calculated as follows:

X 100%

Third, the experimental results:

As can be seen from Figure 9, when cisplatin is used in combination with the peptide of SEQ ID N0.1, their inhibitory effect on A549 cells is significantly stronger than that of cisplatin alone, indicating the peptide of SEQ ID N0.1. It can increase the sensitivity of cisplatin to A549 cells and enhance its inhibitory effect on A549 cells. Experimental Example 7 The effect of SEQ ID NO. 1 peptide, SEQ ID N0.2 peptide and cisplatin on human colon cancer HCT-116 cells was detected by MTT assay.

1. Experimental sample: The peptide of SEQ ID NO. 1 synthesized in Example 2, the peptide of SEQ ID NO. 2 synthesized in Example 3;

Second, the experimental method

The human colon cancer cell line HCT-116 (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) was suspended in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, and seeded in a 96-well plate at a density of 5000-10000 cells/well. After 24 hours of culture, the control group was added with cisplatin, and the experimental group was added a mixture of cisplatin and the peptide of SEQ ID NO. 1, or a mixture of cisplatin and the peptide of SEQ ID NO. The concentration of cisplatin in the control group and the experimental group was 90μιηο1/ί, 30μιηο1/ί, ΙΟμιηοΙ/L, 3.3 mol/L, 1.1μιηο1/ί, 0.37μηιο1/ί, SEQ ID NO. 1 The peptide or the final concentration of the peptide of SEQ ID NO. 1 is 20 μηη _Ο 1 / ί, and 3 replicate wells are set for each concentration. After 20 hours of incubation, MTT (5 mg/ml) 20 μl was added to each well. After 4 hours, the culture solution was aspirated, and DMSO 150 μl was added to each well, and the purple crystals were sufficiently dissolved by shaking for 10 minutes. The absorbance was measured at 570 nm by a microplate reader.

The cell inhibition rate is calculated as follows:

X 100%

Third, the experimental results:

As can be seen from Figure 10, when cisplatin is used in combination with SEQ ID NO. 1, their inhibitory effect on HCT-116 cells is significantly stronger than that of cisplatin alone, indicating that SEQ ID NO. The sensitivity of ammonia platinum to HCT-116 cells enhances its inhibitory effect on HCT-116 cells.

As can be seen from Figure 11, when cisplatin is used in combination with SEQ ID NO. 2, their inhibitory effect on HCT-116 cells is significantly stronger than that of cisplatin alone, indicating that SEQ ID NO. 2 can increase cisplatin. The sensitivity of ammonia platinum to HCT-116 cells enhances its inhibitory effect on HCT-116 cells.

In terms of apoptosis, IC ₅ o can be understood as a certain concentration of a drug to induce apoptosis of tumor cells by 50%, which is called 50% inhibition concentration, that is, when the ratio of apoptotic cells to total cells is equal to 50%. The concentration, IC50 value can be used to measure the ability of the drug to induce apoptosis, that is, the stronger the inducing ability, the lower the value, of course, can also reverse the tolerance of a certain cell to the drug. The results of the combination of SEQ ID NO. 1 and cisplatin on human tumor cells are shown in Table 1. SEQ ID N0.1 and cisplatin are combined. The inhibition of tumor cells is remarkable, and the peptide of SEQ ID N0.1 alone or in combination with cisplatin has a relatively small effect on normal cells, indicating that the peptide of the present invention can be used as a drug for treating and preventing cancer.

Table 1 SEQ ID NO. 1 half-inhibitory concentration (IC ₅ .)

Experimental Example 8 In vivo half-lethal lethal dose (LD50) of the peptide of SEQ ID NO.

1. Experimental sample: Example 2 synthesized SEQ ID NO. 1 peptide;

2. Experimental method: The experiment used 18-22 g of BALB/c mice and males (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). The peptides of SEQ ID N0.1 were set in four dose groups of 125 mg/kg, 250 mg/kg, 500 mg/kg, and 1000 mg/kg, once intraperitoneally, and ended 30 days after administration, weighing, statistical Animals only survive.

III. Experimental results: SEQ ID NO. 1 peptides 125 mg/kg, 250 mg/kg, 500 mg/kg, 1000 mg/kg in four dose groups, 5 animals in each group, administered once intraperitoneally, administered Animals survived when the experiment was terminated in the last 30 days. The experimental results are shown in Table 2. The experimental results demonstrate that the peptide of SEQ ID NO. 1 has an LD50 of >1000 mg/kg.

SEQ ID NO. 1 peptide LD50 (LD50) observation

Dosage route x number of animals weight change (g)

Group

(mg/kg) number of administrations start end start end

125 ipxl 5 5

250 ipxl 5 5

SEQ ID NO. 1 peptide

500 ipxl 5 5

1000 ipxl 5 5

Experimental Example 9 In vivo antitumor effect test of the peptide of SEQ ID NO.

1. Experimental sample: Example 2 synthesized SEQ ID NO. 1 peptide;

Second, the experimental method: Experimental use of 18-22 grams of BALB / c male mice (purchased in Beijing Vitalli Hua Experimental Animal Technology Co., Ltd.). Tumor tissues of colon cancer 26-cell tumor-bearing mice (purchased by the Institute of Biotechnology, Chinese Academy of Medical Sciences) and 0.85% normal saline were ground into a cell suspension at 1:10, and aseptically operated to inoculate tumors under the skin of the right axilla of the animals, each Animals were inoculated with 0.2 ml. The tumor was inoculated for 24 hours. The peptide of SEQ ID NO. 1 was intraperitoneally administered once a day for 10 times. Cisplatin was administered intraperitoneally, once every other day for 5 times; SEQ ID NO. 1 peptide and cisplatin were dissolved in 0.85% physiological saline, and the intraperitoneal dose was 0.2 ml / 20 g body weight. The experiment was performed with a control group (0.85% normal saline), a different dose group of SEQ ID N0.1 peptide, a cisplatin group and a different dose of the peptide of SEQ ID NO. 1 in combination with cisplatin. The experiment was terminated 11 days after the tumor was inoculated, the body weight was weighed, the animals were sacrificed, the tumor was removed, and the weight was weighed. The tumor inhibition rate was calculated as follows, and statistically processed to determine whether there was a significant difference.

Tumor inhibition rate = (average tumor weight of the control group - mean tumor weight of the treatment group) / mean tumor weight of the control group X 100% evaluation of drug combination

The two-drug combination index (CI) CI=AB/(AX B) ₀ AB is the T/C ratio of the two-drug combination group, and A and B are the T/C ratios of the drugs alone (in theory, when C 1 The two drugs have synergistic effects).

Third, the experimental results:

The combination of the peptide of SEQ ID NO. 1 and cisplatin showed a dose-dependent tumor suppressive effect on colon cancer 26 tumor tissue. The peptide doses of SEQ ID NO. 1 were 25 mg/kg, 50 mg/kg, 100 mg/kg, and the combination of cisplatin and lmg/kg had different degrees of antitumor effect. The tumor inhibition rate of SEQ ID NO. 1 peptide 25 mg/kg, 50 mg/kg, 100 mg/kg was 0%, 0% and 4%, respectively; the inhibition rate of cisplatin dose 1 mg/kg was twenty two%. The dose of SEQ ID NO. 1 peptide was 25 mg/kg, 50 mg/kg, 100 mg/kg and cisplatin 1 mg/kg, respectively. The tumor inhibition rate was 23% (P<0.05), 30% and 34% (P < 0.01); the two drug combination index (CI) were 0.99, 0.90 and 0.88, respectively, showing synergy.

In vivo test results showed that the combination of SEQ ID NO. 1 peptide and cisplatin inhibited dose-dependent inhibition of mouse transplanted colon cancer 26 tumor growth, and the combination of SEQ ID NO. 1 peptide and cisplatin showed synergy. effect. Effect of SEQ ID NO.1 peptide combined with cisplatin on colon cancer of mice26

Number of animals Weight change (g)

Route of administration > Χ ± · Inhibition rate Group (mg/k Start End Start End Value CI value Number of doses (g) g)

Control ip> o 10 10 21.32 20.89 1.65±0.32 -- -- cisplatin 1 ip ^; < 5 10 10 21.76 19.73 1.28±0.32 22 <0.05

SEQ ID NO. 1 peptide 25 ip> ao 10 10 21.67 19.98 2.02±0.57 0 >0.05

50 ip> ao 10 10 22.27 19.34 1.97±0.43 0 >0.05

100 ip> ao 10 10 21.50 19.73 1.58±0.22 4 >0.05

SEQ ID NO. 1 peptide 25+1 ipxlO+ ipx 5 10 10 21.80 18.44 1.27±0.40 23 <0.05 0.99

+cis cisplatin

50+1 ipxlO+ ipx 5 10 10 22.70 19.12 1.15±0.36 30 <0.01 0.90 100+1 ipxlO+ ipx 5 10 10 22.10 19.09 1.09±0.30 34 <0.01 0.88

Claims

Claim

A polypeptide having a killing tumor cell effect, characterized in that the amino acid sequence of the polypeptide is represented by SEQ ID NO.

3. A nucleotide sequence encoding the polypeptide of claim 1 or 2.

An expression vector comprising: the nucleotide sequence of claim 3 containing at least one copy.

5. A host cell comprising the expression vector of claim 4.

6. A product obtained by conjugating or mixing a polypeptide according to claim 1 or 2 with a preparation which increases the accumulation of the peptide in a cell.

7. A product according to claim 6 wherein: said formulation is a carrier which assists in the penetration of the peptide into the cell membrane.

8. A product as claimed in claim 7, wherein: the carrier to help the peptide to penetrate the cell membrane of any one of arginine-rich peptide is selected from the following: HIV ₄₈ - ₅₇ peptides, FHV- outer It is ₃₅ - ₄₉ peptides, HTLV-I I Rex ₄ - or too BMV gag ₇ - ₂₅ peptide.

9. A product according to claim 6 wherein: the formulation is selected from the group consisting of nanomaterials, liposomes or oily compounds; or a mixture of a plurality of oily compounds.

A pharmaceutical composition for treating or preventing cancer, which comprises: a therapeutically or prophylactically effective amount of the polypeptide of claim 1 or 2 and a pharmaceutically acceptable adjuvant or carrier.

11. Use of a polypeptide according to claim 1 or 2 for the manufacture of a medicament for the treatment or prevention of cancer.

12. Use of the product of claim 6 in the manufacture of a medicament for the treatment or prevention of cancer.

The use according to claim 11 or 12, wherein the cancer is selected from the group consisting of lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, esophageal cancer, breast cancer, leukemia, bladder cancer, cervical cancer or nose. Pharyngeal cancer.

14. Use of a polypeptide according to claim 1 or 2 for the manufacture of a medicament for enhancing the selective killing of a cancer cell by a genetic toxin.

The use according to claim 14, wherein: the genetic toxin is selected from the group consisting of cisplatin, Oxaliplatin, paclitaxel, epirubicin, doxorubicin, pirarubicin, daunorubicin, mitomycin, dacarbazine, cyclophosphamide, gemcitabine or capecitabine.

The use according to claim 14, wherein the cancer cell is selected from the group consisting of lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, esophageal cancer, breast cancer, leukemia, bladder cancer, cervical cancer or nasopharynx. Cancerous cancer cells.