A composition containing a polypeptide and SOD and its use in treating tumors

Abstract

This invention provides a composition containing a polypeptide and SOD and its use in the treatment of tumors. This invention mixes a novel polypeptide with SOD in a specific ratio, which can induce the death of colorectal cancer cells while being essentially non-toxic to normal cells and significantly inhibiting the growth of tumor cells, providing a new strategy for the preparation of drugs for treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to a composition containing polypeptides and SOD and its use in the treatment of tumors. Background Technology

[0002] Superoxide dismutase (SOD) is an important antioxidant enzyme whose main function is to catalyze the reaction of superoxide anions (O2-). - Superoxide (SOD) is converted into oxygen and hydrogen peroxide, thereby protecting cells from oxidative stress damage. SOD plays a crucial role in the antioxidant defense system of organisms.

[0003] Superoxide anions are harmful byproducts produced during metabolism, which can cause oxidative stress and damage cell membranes, DNA, and proteins. SOD (superoxide dismutase) converts superoxide anions (O2) into superoxide anions (O2) through a two-step reaction. - Superoxide anions are converted into oxygen (O2) and hydrogen peroxide (H2O2), the latter of which can then be further broken down into water by catalase or glutathione peroxidase. By scavenging these superoxide anions, SOD effectively prevents oxidative damage and maintains cell health.

[0004] Superoxide dismutase (SOD) is widely distributed in many tissues and plays a crucial role in protecting cells from oxidative stress and maintaining cellular homeostasis. Studies have shown that SOD activity is closely related to the occurrence and development of various diseases, such as neurodegenerative diseases, inflammatory diseases, cancer, and cardiovascular diseases. Enhancing in vivo SOD activity or supplementing with exogenous SOD may help prevent and treat these diseases. SOD and its analogues have been investigated for drug development and treatment of various diseases associated with oxidative stress.

[0005] Superoxide dismutase (SOD) is a key antioxidant enzyme that protects cells from oxidative stress by catalyzing the conversion of superoxide anions into oxygen and hydrogen peroxide. SOD plays an important biological role in anti-aging, disease prevention and treatment, and maintaining cellular health.

[0006] Peptides are chain-like molecules composed of amino acids linked by peptide bonds, falling between amino acids and proteins. Based on the number of amino acid residues, peptides are generally classified into oligopeptides (less than 10 amino acids), polypeptides (10-50 amino acids), and proteins (more than 50 amino acids).

[0007] Polypeptide hormones, such as insulin and thyroid-stimulating hormone, transmit signals through the bloodstream to regulate various physiological processes in the body. Polypeptides, such as enkephalins and endorphins, play important signal transduction roles in the nervous system. Naturally occurring peptides possess antibacterial, antiviral, and antifungal activities, such as defensins and cathelicidins. Many enzymes are composed of polypeptide chains and catalyze various biochemical reactions. Polypeptides constitute structural proteins, such as keratin and collagen, providing structural support for cells and tissues.

[0008] Synthesizing polypeptide chains by stepwise addition of protecting amino acids is a common method for polypeptide synthesis in the laboratory. Stepwise synthesis of polypeptides in the liquid phase is suitable for the preparation of short peptides. Target polypeptides can be expressed and extracted in cells using genetic engineering techniques. Polypeptides can be chemically modified, such as through lipolysis, glycosylation, and phosphorylation, to enhance their stability, activity, or targeting.

[0009] Peptide drugs have shown broad application prospects in the treatment of cancer, diabetes, and cardiovascular diseases. Peptide vaccines guide the immune system to recognize and attack pathogens or tumor cells, exhibiting high specificity and safety. Peptides have important applications in biomarker detection and disease diagnosis, such as peptide antibody detection. As dietary supplements, peptides are used to enhance immunity, provide antioxidant effects, and combat fatigue. Peptides are used in the preparation of biomaterials, nanomaterials, and smart materials, demonstrating good biocompatibility and controllability.

[0010] Due to their unique structure and function, peptides have broad application prospects in fields such as biology, medicine, and materials science. Through continuous research and technological advancements, the potential of peptides will be further explored and applied, driving scientific and technological progress.

[0011] Colorectal cancer (CRC) is a common malignant tumor of the digestive tract, occurring on the lining of the colon or rectum. It is the third most common cancer and the fourth leading cause of cancer-related death worldwide. Its incidence and mortality rates are higher in developed countries, but with changing lifestyles, the incidence is also rising in developing countries. It is typically more common in people over 50 years of age, with a slightly higher incidence in men than women.

[0012] Familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) are major genetic risk factors. Individuals with a family history of colorectal cancer have a higher risk. A high-fat, low-fiber diet increases the risk. Consumption of red and processed meats is also associated with incidence. Obesity and lack of physical activity increase the risk. Long-term smoking and heavy alcohol consumption are risk factors for colorectal cancer. Chronic inflammatory bowel diseases such as Crohn's disease and ulcerative colitis increase the risk of colorectal cancer.

[0013] Early-stage rectal or colon cancer often presents with no obvious symptoms, or only mild indigestion, bloating, or other discomfort. Late-stage rectal or colon cancer often causes rectal bleeding or darkening of the stool. Other symptoms include changes in bowel habits, such as diarrhea, constipation, or changes in stool shape. Persistent abdominal pain or discomfort may also occur. Cancer progression can lead to weight loss and persistent fatigue.

[0014] Early-stage colorectal cancer is often treated with surgical resection, including local and radical resection. Radiation therapy is used for adjuvant therapy after surgery or systemic treatment of advanced cancer. Commonly used drugs include fluorouracil, oxaliplatin, and irinotecan. Preoperative or postoperative radiotherapy can reduce the risk of recurrence. Targeted therapies such as bevacizumab, Sanofi, and cetuximab are used to treat specific types of colorectal cancer. In recent years, immune checkpoint inhibitors such as pembrolizumab (Keytruda) and nivolumab (Opdivo) have shown potential in treating advanced or metastatic colorectal cancer.

[0015] Colorectal cancer is a common and potentially fatal malignant tumor, but many cases can be effectively controlled or even cured through early screening, lifestyle modifications, and appropriate treatment. Understanding its risk factors, symptoms, diagnosis, and treatment methods is crucial for the prevention and treatment of colorectal cancer. Invention Overview

[0016] To address the problems existing in the prior art, this invention provides a composition containing polypeptides and SOD, a method for preparing the same, and their application in the treatment of colorectal cancer.

[0017] To address this, the present invention adopts the following technical solution:

[0018] In one aspect, the present invention relates to an antitumor composition, characterized in that the antitumor composition comprises an antitumor polypeptide and SOD.

[0019] Furthermore, the antitumor composition of the present invention consists of 10-200 μg / mL of antitumor peptides and 100-1000 U / mL of SOD.

[0020] Furthermore, the antitumor composition of the present invention consists of 10 μg / mL of antitumor polypeptide and 500 U / mL of SOD.

[0021] On the other hand, the antitumor polypeptides described in this invention are selected from:

[0022] (1) A polypeptide having the amino acid sequence shown in KTWFYLQAGHVDSPMNRELC (SEQ ID No. 1); and / or

[0023] (2) A polypeptide that has at least 70% homology with the amino acid sequence shown in SEQ ID No.1 and has the same or similar function.

[0024] Furthermore, the antitumor polypeptide of the present invention is a polypeptide with at least 80%, 85%, 90%, 95%, 97%, 98% or 99% homology to the amino acid sequence shown in SEQ ID No. 1, and having the same or similar functions.

[0025] Furthermore, the amino acid sequence of the antitumor polypeptide is KTWFYLQAGHVDSPMNRELC (SEQ ID No. 1).

[0026] Thirdly, the method for preparing the antitumor polypeptide of the present invention includes the step of synthesizing the antitumor polypeptide using a solid-phase synthesis method.

[0027] Furthermore, the preparation method of the present invention is characterized by comprising the following steps:

[0028] (1) Resin swelling

[0029] RinkAmide AM resin (200-400 mesh), swell with DMF or DCM for 10-30 minutes, then wash with DMF;

[0030] (2) Deprotection

[0031] Treat the Fmoc protecting group on the resin with piperidine / DMF solution, and wash the resin multiple times with DMF or DCM.

[0032] (3) Loading the first amino acid

[0033] The first amino acid is mixed with the activating and conjugating reagents and added to the swollen resin. The mixture is stirred for 1-2 hours to remove the Fmoc group by washing with piperidine / DMF solution for 20-30 minutes. The resin is then washed multiple times with DMF / DCM to remove unreacted reagents and byproducts.

[0034] (4) Gradual elongation of amino acids

[0035] The next amino acid is mixed with the activating agent and the conjugating agent and added to the resin for coupling reaction. The reaction time is 1-2 hours. After each amino acid is coupled, the deprotection step and washing step are repeated. Each amino acid in SEQ ID No. 1 is added in sequence, and the above steps are repeated until all amino acids are synthesized.

[0036] (5) Final deprotection and pyrolysis

[0037] After the last amino acid was synthesized, the final deprotection was performed with piperidine / DMF solution. The resin was washed multiple times with DMF / DCM, and the resin was treated with lysis buffer to lyse the peptide and remove the side chain protecting groups. The reaction time was 1-3 hours. The lysis buffer was collected, the solvent was removed under vacuum, and the solution was lyophilized.

[0038] (6) Chromatography and purification

[0039] The synthesized peptides were purified using instruments, and the purity and molecular weight of the peptides were confirmed using relevant instruments.

[0040] Fourthly, the present invention relates to the use of the antitumor composition described herein in the preparation of a medicament for treating tumors.

[0041] Fifthly, the present invention relates to the use of the antitumor composition described herein in inducing programmed necrosis of cancer cells.

[0042] Sixthly, the present invention relates to the use of the antitumor composition described herein in the preparation of a programmed necrosis inducer for cancer cells.

[0043] Furthermore, the tumor described in this invention is colorectal cancer.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] The novel polypeptide provided by this invention, when mixed with SOD in a specific ratio, can induce the death of colorectal cancer cells, while being essentially non-toxic to normal cells and significantly inhibiting the growth of tumor cells, thus providing a new strategy for the preparation of drugs for treating tumors. Attached Figure Description

[0046] Figure 1 Effects of SOD, PTS, and SOD+PTS combinations on different cell activities

[0047] Figure 2 Caspase 3 / 7 activity in different cells

[0048] Figure 3 Effects of Nec-1s on cell viability Detailed Implementation

[0049] The present invention will be further illustrated below with reference to specific embodiments. Unless otherwise specified, the raw materials, reagents or devices used in the following embodiments can be obtained from conventional commercial sources or by existing known methods.

[0050] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0051] Example 1: Synthesis of Polypeptides

[0052] The preparation method of the antitumor polypeptide KTWFYLQAGHVDSPMNRELC (SEQ ID No. 1) is as follows:

[0053] (1) Resin swelling

[0054] Weigh RinkAmide AM resin (200-400 mesh), add DMF to swell for 15 minutes, and wash with DMF;

[0055] (2) Deprotection

[0056] The Fmoc protecting groups on the resin were treated with a piperidine / DMF (v / v 2:8) solution for 5 minutes twice to remove the Fmoc groups. The resin was then washed with DMF multiple times.

[0057] (3) Loading the first amino acid

[0058] The first amino acid (Fmoc-Cys, 2 equivalents) was mixed with the activating agent (HATU, 2 equivalents) and the conjugating agent (DIPEA, 4 equivalents) and added to the swollen resin. The mixture was stirred at room temperature for 2 hours. The resin was then washed with piperidine / DMF (v / v 2:8) solution for 20 minutes to remove the Fmoc group. The resin was then washed multiple times with DMF to remove unreacted reagents and byproducts.

[0059] (4) Gradual elongation of amino acids

[0060] The next amino acid (Fmoc-Leu, 2 equivalents) was mixed with the activating agent (HATU, 2 equivalents) and the conjugating agent (DIPEA, 4 equivalents), and added to the resin for coupling reaction. The reaction time was 2 hours. After each amino acid was coupled, the deprotection and washing steps were repeated. Each amino acid in SEQ ID No. 1 was added in sequence, and the above steps were repeated until all amino acids were synthesized.

[0061] (5) Final deprotection and pyrolysis

[0062] After the synthesis of the last amino acid, the final deprotection was performed with piperidine / DMF (v / v 2:8) solution. The resin was washed multiple times with DMF and treated with lysis buffer (TFA:TIPS:water = 95:5:5) at room temperature for 2 hours to lyse the peptide and remove the side chain protecting groups. The lysis mixture was collected, the solvent was removed under vacuum, and the crude peptide was dissolved in 0.1% TFA aqueous solution and lyophilized.

[0063] (6) Chromatography and purification

[0064] The synthesized peptides were purified by HPLC, the relevant fractions were collected, lyophilized, and analyzed by LC-MS, MALDI-TOF MS and analytical HPLC.

[0065] Example 2: In vitro antitumor activity test

[0066] 1. Experimental Methods

[0067] MTT method:

[0068] Human normal hepatocytes LO2 were cultured in RPMI-1640 medium (containing 10% FBS and 1% PS) at 5% CO2 and 37°C.

[0069] Human colon cancer cells HCT116 and HT29 were cultured in McCoy's 5A medium (containing 10% FBS and 1% PS) at 5% CO2 and 37°C.

[0070] Cells in the logarithmic growth phase (LO2, HCT116, HT29) were collected, and the cell suspension concentration was adjusted. The cell suspension was seeded into 96-well plates, and the cell density was adjusted to approximately 10,000 cells / well, with 100 μL of cell suspension per well. The plates were incubated at 37°C with 5% CO2 for 24 h. Microscopic observation showed adherent cell growth. SOD, SEQ ID No. 1 peptide, and SOD+peptide combination were added to the culture plates according to the settings in Table 1. PBS was added as a blank control. The culture plates were placed in a 37°C incubator with 5% CO2. After 24 hours, the plates were removed, and 10 μL of MTT (5 mg / mL) solution was added to each well. Culture was continued for 4 h, and the culture was terminated. The culture medium in the wells was carefully aspirated. Then, 100 μL of DMSO was added to each well, and the plates were shaken slowly for 10 min to fully dissolve the purple crystals. The absorbance of each well was measured at 490 nm using an automated microplate reader.

[0071] Table 1 Experimental group settings

[0072]

[0073] In Table 1, PTS represents the polypeptide SEQ ID No. 1; the values ​​in each experimental group represent the final concentration tested.

[0074] 2. Experimental Results

[0075] Experimental results are as follows Figure 1 As shown.

[0076] The results showed that the concentrations of SOD, SEQ ID No. 1 peptide, and SOD+peptide composition used in this invention were essentially non-toxic to normal human hepatocytes LO2. SOD and SEQ ID No. 1 peptide alone had no significant inhibitory effect on HCT116 and HT29 human colon cancer cells, while the SOD+peptide composition showed significant inhibitory activity against HCT116 and HT29 human colon cancer cells. Furthermore, the combination of 500 U / mL SOD and 10 μg / mL PTS showed the most significant inhibitory effect on cancer cells.

[0077] Example 2: Investigation into the antitumor mechanism of the composition

[0078] Caspase 3 / 7 method:

[0079] Human colon cancer cells HCT116 and HT29 were cultured in McCoy's 5A medium (containing 10% FBS and 1% PS) at 5% CO2 and 37°C.

[0080] Cells in the logarithmic growth phase (HCT116, HT29) were collected, and the cell suspension concentration was adjusted. The cell suspension was seeded into 96-well plates, with a cell density adjusted to approximately 10,000 cells / well (100 μL of cell suspension per well). The plates were incubated at 37°C with 5% CO2 for 24 hours. Microscopic observation showed adherent cell growth. 10 μL of an SOD+PTS combination (final SOD concentration 500 U / mL, final PTS concentration 10 μg / mL) was added to each well. PBS was added as a blank control, and cisplatin (cis) as a positive control. The plates were incubated in a 37°C incubator with 5% CO2. After 24 hours, the plates were removed, and Caspase 3 / 7 activity was measured using a Caspase-Glo assay kit (Promega, Madison, Wisconsin, USA). Caspase 3 / 7 activity was then calculated according to the manufacturer's instructions.

[0081] Experimental results are as follows Figure 2 As shown.

[0082] The experimental results showed that CIS treatment significantly enhanced the Caspase 3 / 7 activity in HCT116 and HT29 cells, indicating that CIS can induce apoptosis in cancer cells. However, the Caspase 3 / 7 activity in cells treated with the SOD+PTS combination was not significantly enhanced, suggesting that the SOD+PTS combination does not exert its antitumor activity by inducing apoptosis in cancer cells.

[0083] Further investigation is needed to determine whether the SOD+PTS combination generates antitumor activity by inducing other types of cell death.

[0084] MTT method:

[0085] Human colon cancer cells HCT116 and HT29 were cultured in McCoy's 5A medium (containing 10% FBS and 1% PS) at 5% CO2 and 37°C.

[0086] Cells in the logarithmic growth phase (HCT116, HT29) were collected, and the cell suspension concentration was adjusted. The cell suspension was seeded into 96-well plates, with a cell density adjusted to approximately 10,000 cells / well (100 μL of cell suspension per well). The plates were incubated at 37°C with 5% CO2 for 24 hours. Microscopic observation showed adherent cell growth. 10 μL of the necroptosis inhibitor Nec-1s was added to the culture plate for pretreatment for 1 hour. After pretreatment, the medium containing the inhibitor was discarded, and fresh medium was added, along with 10 μL of a SOD+PTS combination (final SOD concentration 500 U / mL, final PTS concentration 10 μg / mL). PBS was used as a blank control. The culture plates were placed in a 37°C incubator with 5% CO2. After 24 hours, the plates were removed, and 10 μL of MTT (5 mg / mL) solution was added to each well. Culture was continued for 4 hours, then the culture was terminated, and the culture medium in the wells was carefully aspirated. Next, add 100 μl of DMSO to each well and place on a shaker with low speed for 10 min to fully dissolve the purple crystals. Measure the absorbance of each well at 490 nm using an automated microplate reader.

[0087] Experimental results are as follows Figure 3 As shown.

[0088] The experimental results show that the SOD+PTS combination can induce cell death in HCT116 and HT29 cells, and Nec-1s can significantly reverse the cancer cell death induced by the SOD+PTS combination. Therefore, the SOD+PTS combination kills cancer cells by inducing programmed necrosis.

[0089] In summary, the SOD+PTS composition of this invention can kill cancer cells by inducing programmed necrosis, and has good prospects for clinical application.

[0090] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. An antitumor composition, characterized in that, The antitumor composition is composed of antitumor peptides and SOD; The antitumor composition consists of 10 μg / mL of antitumor peptide and 500 U / mL of SOD; The amino acid sequence of the antitumor polypeptide is SEQ ID No.

1.

2. The antitumor composition according to claim 1, characterized in that, The method for preparing the antitumor polypeptide includes the following steps: (1) Resin swelling 200-400 mesh Rink Amide AM resin, swelled with DMF or DCM for 10-30 minutes, then washed with DMF; (2) Deprotection Treat the Fmoc protecting group on the resin with piperidine / DMF solution, and wash the resin multiple times with DMF or DCM. (3) Loading the first amino acid The first amino acid is mixed with the activating and conjugating reagents and added to the swollen resin. The mixture is stirred for 1-2 hours to remove the Fmoc group by washing with piperidine / DMF solution for 20-30 minutes. The resin is then washed multiple times with DMF / DCM to remove unreacted reagents and byproducts. (4) Gradual elongation of amino acids The next amino acid is mixed with the activating agent and the conjugating agent and added to the resin for coupling reaction. The reaction time is 1-2 hours. After each amino acid is coupled, the deprotection step and washing step are repeated. Each amino acid in SEQ ID No. 1 is added in sequence, and the above steps are repeated until all amino acids are synthesized. (5) Final deprotection and pyrolysis After the last amino acid was synthesized, the final deprotection was performed with piperidine / DMF solution. The resin was washed multiple times with DMF / DCM, and the resin was treated with lysis buffer to lyse the peptide and remove the side chain protecting groups. The reaction time was 1-3 hours. The lysis buffer was collected, the solvent was removed under vacuum, and the solution was lyophilized. (6) Chromatography and purification The synthesized peptides were purified using instruments, and the purity and molecular weight of the peptides were confirmed using relevant instruments.

3. The use of the antitumor composition according to claim 1 or 2 in the preparation of a medicament for treating tumors, wherein, The tumor is colorectal cancer.

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