TW202210094A - Purpose of carnosine nanoparticles for use in preparing medicine of controlling intestinal cancer cells achieves the advantage of suppressing the growth of cancer cells to inhibit the cancer - Google Patents

Abstract

A purpose of carnosine nanoparticles for use in preparing medicine of controlling intestinal cancer cells is used for discussing the influences of suppressing the active mechanism of a cell strain HCT-116 of human colorectal cancer through Carnosine nanoparticles, C-NPs at different concentrations of 0.5, 1, 5, 10, 15 mM. It can be known from an experimental result, the Carnosine nanoparticles can, by controlling apoptosis, autophagy and necrosis/necroptosis of the cell strain HCT-116 mechanisms, further achieve the advantage of suppressing the growth of cancer cells to inhibit the cancer.

Description

Use of carnosine nanoparticles for preparing drugs for regulating intestinal cancer cells

The present invention relates to a kind of carnosine nanoparticle for preparing medicine for regulating intestinal cancer cells The use of the substance, especially involving carnosine nanoparticles (C-NPs), can regulate the apoptosis (Apoptosis), autophagy (Autophagy) and necrosis/programmed necrosis of HCT-116 cell line ( Necrosis/Necroptosis) mechanism to inhibit the growth of cancer cells to inhibit cancer.

According to the World Health Organization, about 9.6 million people died of cancer in 2018. Colon cancer ranks third in the list, and the cancer that causes death ranks second. In 2019, the Ministry of Health and Welfare announced the top ten causes of death in Taiwan in 2018. Malignant tumor (cancer) was the top cause of death, and cancer was the top ten cause of death in Taiwan. Colon cancer ranked third. From this, it is known that colon cancer is a major threat to human health, and the development of drugs to prevent colon cancer has become a current research trend.

Carnosine is composed of β-alanine and L-histidine (L-histidine) is a combination of double peptides, mainly found in vertebrate skeletal muscle, known to prevent nerve and brain degeneration, anti-oxidation and anti-cancer effects. The applicant has investigated the effect of carnosine on inhibiting the formation of colon cancer induced by Azoxymethane (AOM), and found that carnosine can reduce the number of intestinal tumors, abnormal crypt (AC) and abnormal crypt foci (ACF) by reducing the number of intestinal tumors. ) and the expression of genes and proteins of terminal homeobox protein 2 (CDX2) and cytokeratin (Krt20), which are specific markers of colon cancer, to inhibit the formation of colon cancer.

Due to the unique physical properties of nanoparticles, they have received special attention in medical-related fields. It is often used in biomedical diagnostic imaging and drug development. For this reason, it is necessary to further develop a set of carnosine nanoparticles that can inhibit the growth of human colon cancer HCT-116 cell line and improve the aforementioned shortcomings of the prior art.

The main purpose of the present invention is to provide a carnosine nanoparticle that can be controlled by Apoptosis, autophagy and necrosis/programmed necrosis mechanism of HCT-116 cell line can inhibit the growth of cancer cells to inhibit cancer.

；In order to achieve the above purpose, the present invention relates to the use of carnosine nanoparticles for preparing a drug for regulating intestinal cancer cells, comprising administering an effective dose of a compound or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier ; The compound is carnosine nanoparticles with a molecular weight of 453.2 and a density of 1.019. Its structural formula is as follows:

;

In the above embodiments of the present invention, the carnosine used in the carnosine nanoparticles is (2S)-2-[(3-Amino-1-oxopropyl)amino]-3-(3H-imidazol-4-yl)propanoic acid with a molecular weight of 226.23 and a density of 1.4.

Wherein, the carnosine nanoparticles were administered at a dose concentration of 0.5 to 15 mM, and the colorectal cancer cells were treated with It is a human colon cancer HCT-116 cell line. The dosage form of the drug is a solution, which is mixed with the culture medium. It is a human colon cancer HCT-116 cell line. The dosage form of the drug is a solution, and the HCT-116 cell line is treated by mixing with the culture medium, and the cell treatment time is 72 or 96 hours.

Please refer to "Figure 1 to Figure 7C", which are respectively the carnosine nanoparticles of the present invention Schematic diagram of the experimental structure for the study of the mechanism of the particles on the inhibition of human colon cancer HCT-116 cell line, the cell morphology of the carnosine nanoparticles of the present invention treated HCT-116 cell line for 72 or 96 hours, the carnosine nanoparticles of the present invention on HCT Schematic diagram of changes in cell viability of -116 cell line, schematic diagram of changes of carnosine nanoparticles of the present invention on apoptosis of HCT-116 cell line, changes of Bax gene expression of HCT-116 cell line by carnosine nanoparticles of the present invention Schematic diagram, the change diagram of carnosine nanoparticles of the present invention on the expression level of Bcl 2 gene in HCT-116 cell line, the carnosine nanoparticles of the present invention on the Caspase 9 gene of HCT-116 cell line Schematic diagram of the change of expression amount, the change diagram of carnosine nanoparticles of the present invention to the expression of Caspase 3 gene in HCT-116 cell line, and the schematic diagram of the change of the expression amount of carnosine nanoparticles of the present invention to Caspase 8 gene of HCT-116 cell line , Schematic diagram of the change of carnosine nanoparticles of the present invention on the expression of PARP gene in HCT-116 cell line, schematic diagram of the change of carnosine nanoparticles of the present invention on autophagy of HCT-116 cell line cells, and carnosine nanoparticles of the present invention. Schematic diagram of the change of the expression level of Beclin 1 gene in HCT-116 cell line, schematic diagram of the change of the expression level of PI3K III gene of HCT-116 cell line by carnosine nanoparticles of the present invention, and the change of carnosine nanoparticles of the present invention on HCT-116 cell line LC 3. Schematic diagram of changes in gene expression, schematic diagrams of changes in ROS content of HCT-116 cell lines by carnosine nanoparticles of the present invention, schematic diagrams of changes in ATP content of HCT-116 cell lines by carnosine nanoparticles of the present invention, and Schematic diagram of the changes of carnosine nanoparticles on the expression of MLKL gene in HCT-116 cell line.

As shown in the figure: the present invention uses carnosine nanoparticles as experimental materials, and uses human Colon cancer HCT-116 cell line was treated with 0.5, 1, 5, 10, 15 mM carnosine nanoparticles for 72 or 96 hours, and sterilized water was used as a control group. effects on cell viability. Then 96 hours were selected for follow-up experiments, and apoptosis was analyzed by Annexin V (Annexin V)/Propidium Iodide (PI) staining and its related indicators Bcl 2 associated protein (Bcl 2 associated X, Bax), B cell lymphoma (Bcl 2), cysteine protease 3, 8, 9 (Caspase 3, 8, 9), and ADP-ribose polymerase (Poly (ADP-ribose) polymerase, PARP) gene expression The amount of autophagy; autophagy test and related indicators phosphoinositide 3 kinase III (Phosphoinositide 3 kinase, PI3K III), autophagy factor (Beclin 1) and autophagy microtubule-related protein 1B light chain 3 (Microtubule- associated protein 1B light chain 3, LC3) gene expression; and cell necrosis/programmed necrosis by reactive oxygen species (ROS) and adenosine triphosphate (ATP) content analysis and mixed lineage kinase protein (Mixed lineage) kinase domain like protein, MLKL) gene expression, and the anti-cancer mechanism of carnosine nanoparticles was explored through the above experiments. The experimental structure is shown in Figure 1. To evaluate whether carnosine nanoparticles can inhibit the growth of human colon cancer HCT-116 cell line, and further explore whether the inhibition mechanism is regulated by apoptosis, autophagy and necrosis/programmed necrosis.

The invention relates to the use of carnosine nanoparticle for preparing a drug for regulating intestinal cancer cells In this way, the provided compounds or their pharmaceutically acceptable salts can be combined with the compounds provided in this case or their pharmaceutically acceptable salts, and at least one medicinal The above acceptable carrier is used to prepare a dosage form suitable for the medicament of the present invention, and the medicament can be administered to a subject by intravenous injection, subcutaneous injection, oral administration, or application. The following examples are only examples for understanding the details and connotations of the present invention, but are not intended to limit the scope of the patent application of the present invention.

The above mentioned carriers include but are not limited to excipients, diluents, thickeners, fillers, Binder, disintegrant, lubricant, oily or non-oily base, surfactant, suspending agent, gelling agent, adjuvant, preservative, antioxidant, stabilizer, color, or flavor.

The above mentioned dosage forms include but are not limited to solutions, emulsions, suspensions, powders, lozenges, Oils, ointments, lozenges, or capsules and other similar or suitable dosage forms of the present invention.

[Example 1] Experiment material carnosine and carnosine nanoparticle sources Carnosine ((2S)-2-[(3-Amino-1-oxopropyl)amino]-3-(3H-imidazol-4-yl ) propanoic acid, Carnosine), purchased from Sigma Company (Sigma-Aldrich Co. USA); carnosine nanoparticles were provided by Professor Xie Shuzhen, Aerosol Science Research Center, National Sun Yat-Sen University. The molecular weight of the carnosine is 226.23, and the density is 1.4; the molecular weight of the carnosine nanoparticle is 453.2, and the density is 1.019. The structural formula of the carnosine nanoparticle is as follows:

[Example 2] Experimental method 1. Culture of human colon cancer HCT-116 cell line Penicillin/Streptomycin) 100 U/mL, 2.2 g/L sodium bicarbonate (Sodium bicarbonate), and 10% Fetal bovine serum (FBS) in McCoy's 5A medium, placed in 5% carbon dioxide (CO ₂ ) in a constant temperature incubator at 37°C.

2. Observation of cell morphology 3×10 ⁵ cell/mL HCT-116 cell line was planted in a 3.5 cm petri dish, and cultured in a 37°C incubator with 5% CO ₂ until the next day. 0.5, 1, 5, 10 or 15 mM carnosine nanoparticle solution, and the medium containing sterilized water was used as the control group. After culturing for 72 or 96 hours, an inverted phase contrast microscope was used to observe and record the changes in cell morphology.

3. Cell viability test In this experiment, the cell viability test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) was used as the method to detect the viability of HCT-116 cell line . After the HCT-116 cell line was sampled, washed twice with Phosphate buffered saline (PBS), added McCoy's 5A containing 0.5 mg/mL MTT, and placed in a 37°C incubator containing 5% CO ₂ After culturing for 3 hours, the medium was removed, 1 mL of Isopropanol was added to dissolve Formazan, shaken for 15 minutes, shaken, and then taken into a 1.5 mL microcentrifuge tube, and centrifuged at 4°C and 9,560 × g for 10 minutes, and 200 μL of the supernatant was taken to 96-well plate, and its absorbance was detected at a wavelength of 570 nm.

4. Cell cycle test In this experiment, a multi-light source micro cell automatic analyzer (NC-3000 Image cytometry) was used for detection. When cells are in the cell cycle, the content of deoxyribonucleic acid (DNA) will increase or decrease with different cycles. Using quantitative 4',6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI) (Solution 12) combined with double-stranded DNA and the fluorescence intensity generated, to determine the percentage of each phase of the cell cycle of the sample cell fluid. After sample treatment, HCT-116 cells were washed twice with PBS, added with 100 μL of 0.25% trypsin (Trypsin), placed in a constant temperature incubator containing 5% CO ₂ at 37°C for 3 minutes, and then incubated with 1 mL of medium. Cells were collected in 1.5 mL microcentrifuge tubes, centrifuged at 25°C, 95 x g for 5 minutes, and the supernatant was removed. Add 1 mL of PBS to mix with the cells, centrifuge at 25°C and 95×g for 5 minutes, remove the supernatant, and redissolve the precipitated cells in 300 μL of PBS. About 1×106 cells were taken out, mixed with 100 μL of Solution 10 and 2 μL of Solution 12, dried at 37°C for 5 minutes, added 100 μL of Solution 11 to stop the reaction, loaded into Slide-A8, and used NC-3000 Cycle mode for analysis.

5. Annexin V/PI staining test This experiment uses a multi-light source trace cell automatic analyzer for detection, Annexin V is a calcium ion-dependent automatic analyzer for detection in this experiment, and Annexin V is a calcium ion-dependent Phospholipid-binding protein that binds to the membrane phospholipid phosphatidylserine (PS) on early apoptotic cells. PI interacts with late apoptotic and dying cells. The percentage of apoptotic cells was calculated by quantifying the fluorescence produced by Annexin V and PI double stain. After sample treatment, HCT-116 cells were washed twice with PBS, added with 100 μL of 0.25% Trypsin, placed in a 37°C incubator containing 5% CO ₂ for 3 minutes, and then harvested with 1 mL of medium at 1.5 mL microcentrifuge tubes, centrifuge at 25°C, 95 x g for 5 minutes, and remove the supernatant. Add 100 μL of PBS and mix with the cells, centrifuge at 25°C and 95×g for 5 minutes, and remove the supernatant. Add 100 μL of dye mixture (containing 100 μL Apoptosis binding buffer + 1 μL Annexin V + 1 μL PI), react in the dark for 15 minutes, centrifuge at 25°C, 95×g for 5 minutes, Remove dye. Add 100 μL of PBS and 2 μL of Solution 15 to mix with the cells, dry bath at 37°C for 5 minutes, centrifuge at 25°C for 5 minutes at 95×g, and remove the supernatant. After washing twice with 300 μL PBS, the pelleted cells were redissolved in 300 μL PBS, loaded into Slide-A2, and analyzed using NC-3000 in Annexin V assay (assay) mode.

6. Autophagy Assay In this experiment, the CYTO-ID® Autophagy Commercial Kit 2.0 (ENZ-51031, USA) purchased from Enzo Company was used, and the multi-light source micro-cell automatic analyzer was used for detection. After sample treatment, HCT-116 cells were washed twice with PBS containing 2% FBS, added with 100 μL of 0.25% Trypsin, placed in a 5% CO ₂ incubator at 37°C for 3 minutes, and then used 1 mL containing 2 Cells were collected in 1.5 mL microcentrifuge tubes in PBS with % FBS, centrifuged at 25°C, 860 × g for 5 minutes, the supernatant was removed, and the cells were redissolved in 200 μL of PBS containing 2% FBS. Add 0.4 μL Cyto-ID green stain solution (Green stain solution), dry bath at 37°C for 15 minutes in the dark, add 0.2 μL Hoechst Fluorescent Dye 333 (Hoechst 33342), dry at 37°C in the dark Bath for 15 minutes. Centrifuge at 25°C, 860 × g for 5 minutes, remove the supernatant, wash twice with 100 μL of PBS containing 2% FBS, redissolve the pelleted cells in 100 μL of PBS containing 2% FBS, and load into Slide- A2, Analysis was performed in Autophagy assay-10000 mode using NC-3000.

7. Determination of ROS content In this experiment, a multi-light source microcell automatic analyzer was used for detection, and 2,7-dichlorofluorescin diacetate (DCFH-DA) was used to penetrate the cell membrane and react with intracellular ROS. , to produce 2,7-dichlorofluorescein (DCF) with fluorescent properties, and by quantifying DCF fluorescence, intracellular ROS content was calculated. After HCT-116 cells were sampled, washed twice with PBS, added 1 mL of 10 μM DCFH-DA solution, and placed in a 37°C incubator containing 5% CO ₂ to react for 30 minutes. Washed twice with PBS, added 100 μL of 0.25% Trypsin, placed in a 37°C constant temperature incubator with 5% CO ₂ for 3 minutes, and collected the cells in a 1.5 mL microcentrifuge tube with 1 mL of medium at 25°C. , 95 × g for 5 minutes, and remove the supernatant. Add 1 mL of PBS to mix with the cells, centrifuge at 25°C and 95×g for 5 minutes, remove the supernatant, and redissolve the precipitated cells in 300 μL of PBS. About 1×106 cells were taken out, mixed with 2 μL of Solution 15, dried at 37°C for 5 minutes, centrifuged at 25°C, 95×g for 5 minutes, and the supernatant was removed. After washing twice with 300 μL PBS, the precipitated cells were redissolved in 100 μL PBS, loaded into Slide-A2, and analyzed in ROS-DCF assay mode using NC-3000.

8. ATP Content Determination Deproteinizing sample preparation kit (Deproteinizing sample preparation kit. Model: K808, USA) and ATP content commercial kit (ATP) were used in this experiment. colorimetric/fluorometric assay kit. Model: K354, USA). After the HCT-116 cells were sampled, they were washed twice with PBS, and the cells were gently scraped with 100 μL ATP assay buffer and collected in a 1.5 mL microcentrifuge tube. C. Centrifuge at 16,200×g for 3 minutes, take 80 μL of supernatant and add 16 μL of perchloric acid (PCA) to mix, put on ice for 5 minutes, and centrifuge at 4°C, 16,200×g for 2 minutes. Take 76.8 μL of the supernatant to another microcentrifuge tube, add 3.2 μL of Neutralization solution, put it on ice for 5 minutes, precipitate the excess PCA, and then centrifuge at 4°C, 16,200×g for 2 minutes , and the supernatant is the deproteinized cell sample solution. Take out 0, 2, 4, 6, 8, 10 μL of 1 mM ATP standard (ATP standard) and 15 μL of sample solution to a 96-well plate respectively, and quantify the volume of each well solution to 50 mL with ATP buffer. After adding the reaction reagents containing 44 μL ATP buffer, 2 μL ATP probe (ATP probe), 2 μL ATP converter (ATP converter) and 2 μL developer mix (Developer mix), the reaction was performed at room temperature for 30 minutes in the dark. , using an enzyme immunoassay analyzer (Enzyme-linked Immuno-sorbent Assay reader, ELISA reader) to detect its absorbance at a wavelength of 570 nm.

9. Ribonucleic acid (RNA) extraction Sterilize all utensils at high temperature and autoclave to remove residual ribonuclease (RNase). After HCT-116 cells were sampled, 1 mL of TRIzol reagent (TRIzol reagent) was used to collect the cells into a 1.5 mL microcentrifuge tube. After thorough mixing and shaking, 200 μL of chloroform (Chloroform) was added and shaken up and down to mix, and ice bathed for 5 minutes. Centrifuge at 16,200×g for 15 minutes at 4°C. Transfer the supernatant to another microcentrifuge tube and add an equal amount (1:1) of isopropanol. After shaking and mixing, ice bath for 5 minutes to precipitate RNA. Centrifuge at 16,200 × g for 15 minutes at 4°C, remove the supernatant, and precipitate as RNA. Add 1 mL of 75% alcohol, centrifuge at 4°C, 16,200×g for 10 minutes, remove the upper layer of alcohol, and then centrifuge at 4°C, 16,200×g for 1 minute, remove the remaining alcohol, and air dry at room temperature for 15 minutes , and finally redissolved in diethyl pyrocarbonate deionized water (Diethyl pyrocarbonate water, DEPC-H ₂ O), and stored at -20°C for later use after quantification.

10. Quantitative ribonucleic acid The nucleic acid protein analysis system is used for RNA quantification. Before the measurement, 2 µL of DEPC-H ₂ O must be taken in the sample holding tank for blank correction. After wiping with 75% alcohol, take 2 µL of each sample into the sample holding tank for testing. Repeat the above actions until the sample testing is completed. After the measurement is completed, it must be wiped with 75% alcohol and calculated by computer software (Gen 5). Sample RNA concentration and RNA quality (OD ₂₆₀ /OD ₂₈₀ ).

11. Reverse transcription of ribonucleic acid into complementary deoxyribonucleic acid (Complementary Deoxyribonucleic acid, cDNA) This experiment was carried out according to the method of the Promega manufacturer. In a 1.5 mL microcentrifuge tube, 0.125 μg/μL oligodeoxythymidylate (Oligo Dt) 0.5 μL, 10 mM deoxynucleoside triphosphate (dNTP) 1 μL and RNA 1 μg sample were added , then make up the volume to 20 μL with sterile water, dry bath at 65°C for 5 minutes, and place on ice for 5 minutes. Then add 1 μL M-MLV Reverse transcriptase (M-MLV Reverse transcriptase), 1 μL RNase inhibitor (RNasin) and 5 μL M-MLV RT 5X buffer in sequence, mix all the reagents by shaking, and store at 37°C. Dry bath for 1 hour to obtain cDNA solution and store at -20°C for later use.

12. In the real-time polymerase chain reaction real-time fluorescence quantitative detection system (Real-time Quantitative PCR Detecting System, Real-time PCR), the SYBR green I dye is combined with the double-stranded DNA of the sample, and the fluorescence is excited under a halogen lamp. , when the cycling reaction increases target gene synthesis, the amount of SYBR green I dye binding to double-stranded DNA also increases. Therefore, as the target gene is amplified, more SYBR green I fluorescence can be detected. Real-time PCR mainly uses the different concentrations of each target gene in the sample. Under the same PCR reaction conditions, the reaction of the target gene template with higher content will reach the geometric phase faster, and the DNA synthesis will be twice as fast. to multiply. The critical cycle number in the Geometric phase is defined as the cycle threshold (Cycle threshold, CT). In order to confirm the operation method and the presence/absence of contamination in the reaction process, a group of negative control group (Non-template control, NTC) without gene was added. In order to compare each quantitative result with each other and avoid the deviation caused by the difference of PCR reaction efficiency, more than one internal control group (Internal control) will be added to each quantitative analysis, using specific primers and internal control group reactions to convert the concentration to confirm the accuracy of each quantification. Before performing Real-time PCR quantitative analysis, 2.5 μL of double-distilled water (ddH ₂ O), 5 μL of 2X SYBR Fast MM, 0.5 μL of 20X target primer, and 2 μL of 20X target primer should be added in sequence to a 96-well plate dedicated to PCR. cDNA (10 ng) in a final volume of 10 μL. Put into Real-time PCR (LightCycler® 96 Real-Time PCR System, Roche Life Science, Basel, Switzerland) reaction, Real-time PCR set conditions, as shown in Table 1. Table I program 1 95°C 120 sec × 1 cycle program 2 95°C 5 sec × 40 cycles 60°C 30 sec Procedure 3 95°C 10 sec × 1 cycle 65°C 60 sec 97°C 1 sec Since the relative quantification needs to select genes with constant expression characteristics of humans as the necessary calculation values for normalization, human β-actin (β-actin) is selected as the control group in this experiment, and each gene of Real-time PCR is specific. Sexual primers, as shown in Table 2Table 2 Gene Primer sequence (5-3) Reference (NCBI GenBank) β-actin F-ATGTGCAAGGCCGGCTTC NM001101.3 R-GAATCCTTCTGACCCATGCC Bax F-TGTTTTCTGACGGCAACTTCA NM001291428.1 R-AGCCCATGATGGTTCTGATCA Bcl-2 F-CCTGTGGATGACTGAGTACCTGAAC NM000633.2 R-CAGCCAGGAGAAATCAAACAGA Caspase 3 F-TGGATTATCCTGAGATGGGTTTATG NM004346.3 R-GCTGCATCGACAYCTGTACCA Caspase 8 F-TCCAAATGCAAACTGGATGATG NM001080124.1 R-TTTTCAGGATGCCAACTTTCCTT Caspase 9 F-AGCTGGACGCCATATCTAGTTTG NM001229.4 R-AACGTACCAGGAGCCACTCTTG PARP F-TGGTCAAGACACAGACACCCA NM001618.4 R-ACGGAGGCGCTGGTTTCT MLKL F-CCTGGGCACAGGAAGATCAG NM001142497.2 R-TTTCTAATCGTCTCAGTGAAGCTTCT PI3K III F-TTGGAGACAGGCACCTGGAT NM001308020.1 R-CCATTTCTTTATTCAGCTTCATTGG Beclin-1 F-CTGGACACGAGTTTCAAGATCCT NM001313998.1 R-GTTAGTCTCTTCCTCCTGGGTCTCT Gene Primer sequence (5-3) Reference (NCBI GenBank) LC3 F-TCCTGGACAAGACCAAGTTTTTG NM032514.3 R-ACCATGCTGTGCTGGTTCAC iNOS F-GTGACCCTGAGCTCTTCGAAA NM000625.4 R-GCGTACCACTTTAGCTCCAGTTC COX 2 F-TGTTTGCATTCTTTTGCCCAG NM000963.4 R-TTACGCTGTCTAGCCAGAGTTTTCA PCNA F-GCGCTAGTATTTGAAGCACCAA NM002592.2 R-GTTCAACATCTAAATCCATCAACTTCAT Cyclin D1 F-TGAACACTTCCTCTCCAAAATGC NM053056.2 R-GCGGATTGGAAATGAACTTCAC β-catenin F-TGCTTGTTCGTGCACATCAG NM001098209.1 R-TGTGAAGGGCTCCGGTACA VEGF F-CGAGGGCCTGGAGTGTGT NM001025366.2 R-TGGTGAGGTTTGATCCGCATA EGFR F-GGCGTCCGCAAGTGTAAGAA NM005228.5 R-TCGTAGCATTTATGGAGAGTGAGTCT HIF-1α F-TAACTTTGCTGGCCCCAGC NM001243084.1 R-ACTTCCTCAAGTTGCTGGTCATC

13. Statistical Results The experimental results were analyzed and compared within the group by the computer statistical analysis software SPSS12.0 (SPSS, USA) program One-way ANOVA, and then compared by Tukey's test, there were significant differences. The degree was set as P<0.05.

[Example 3] Experimental results (1) Changes of carnosine nanoparticles on cell viability of HCT-116 cell line The changes of carnosine nanoparticles on cell viability of HCT-116 cell line are shown in Figures 2A and 2B. From the observation of cell morphology at 72 or 96 hours in Figure 2A, it was found that 10 and 15 mM carnosine nanoparticles increased cancer cell filaments. The cell viability results in Figure 2B showed that the cell viability of HCT-116 cell line was significantly lower than that of the control group after 1, 5, 10, 15 mM carnosine nanoparticles were treated for 72 and 96 hours, indicating that 1, 5, 10, 15 mM carnosine nanoparticles inhibited the viability of HCT-116 cell line in a dose-dependent manner. The data were expressed as the mean ± SD of independent experiments, and the significance of differences in activity at different doses was assessed by Tukey's test statistical analysis. Different superscript letters a, b, c and d in Figure 2B indicate significant differences in cell viability (P<0.05). The HCT-116 cell line was treated with 0.5, 1, 5, 10, and 15 mM carnosine nanoparticles for 96 hours as the subsequent experimental conditions.

(2) Changes of carnosine nanoparticles on apoptosis of HCT-116 cell line The changes of the apoptosis ratio of HCT-116 cell line and the expression of related index genes by carnosine nanoparticles were shown in Figures 3 and 4A to 4F. From the cell apoptosis analysis in Figure 3, it was shown that 5, 10 and 15 mM carnosine nanoparticles treated the HCT-116 cell line for 96 hours, and the apoptotic ratio was significantly higher than that of the control group. From the analysis results of apoptosis-related index gene expression in Figures 4A to 4F, it was found that 5, 10, and 15 mM carnosine nanoparticles can significantly increase the expression of Caspase 8, 9 and PARP genes compared with the control group; 10, 15 mM Carnosine nanoparticles can significantly increase the expression of Caspase 3 gene compared with the control group; 15 mM carnosine nanoparticles can significantly increase the expression of Bax gene compared with the control group; the expression of Bcl 2 gene in different doses of carnosine nanoparticles is significantly different from that of the control group. There was no significant difference between groups. Based on the above experimental results, 5, 10, and 15 mM carnosine nanoparticles can induce apoptosis of colon cancer cells by increasing the expression of Bax, Caspase 3, 8, 9 or PARP genes. The data are expressed as the mean ± SD of independent experiments, and the ratio of apoptosis and the expression of Bax, Bcl 2, Caspase 9, 3, 8 or PARP genes were evaluated by Tukey test statistical analysis. , b and c, indicate statistically significant differences (P < 0.05).

(3) Changes in autophagy of HCT-116 cell line by carnosine nanoparticles The changes of autophagy ratio and related index gene expression in HCT-116 cell line by carnosine nanoparticles, the results are shown in Figures 5 and 6A-6C. From the results of autophagy analysis in Figure 5, it was found that 10, 15 mM carnosine nanoparticles treated HCT-116 cell line for 96 hours, the proportion of autophagy was significantly higher than that of the control group. The analysis results of related index gene expression in Figures 6A to 6C showed that the expression of Beclin 1 gene was significantly higher in 5, 10, and 15 mM carnosine nanoparticles than in the control group; The expression level of PI3K III gene was significantly higher than that in the control group; while 1, 5, 10, and 15 mM carnosine nanoparticles made the expression level of LC 3 gene significantly lower than that in the control group. Based on the above results, 10 and 15 mM carnosine nanoparticles can promote autophagy in colon cancer cells by increasing the expression of Beclin 1 and PI3K III genes, but not regulating the expression of LC 3 gene. The data are expressed as the mean ± SD of independent experiments, and the differences in the proportion of autophagy and the expression of Beclin 1, PI3K III or LC 3 genes were evaluated by Tukey's test statistical analysis. Indicates that there is a statistical difference (P < 0.05).

(4) Changes of carnosine nanoparticles on cell necrosis/programmed necrosis of HCT-116 cell line Changes in the contents of necrosis/programmed necrosis-related indexes or gene expression of HCT-116 cell line by carnosine nanoparticles, the results are shown in Figures 7A to 7C. Figures 7A to 7C show that 5, 10, 15 mM carnosine nanoparticles treated HCT-116 cell line for 96 hours, the ATP content was significantly lower than the control group, and the MLKL gene expression level was significantly higher than the control group; 15 mM carnosine nanoparticle Rice particles made the ROS content significantly higher than that of the control group. Based on the above results, 5, 10 and 15 mM carnosine nanoparticles can promote necrosis and programmed necrosis of colon cancer cells by increasing the expression of intracellular ROS and MLKL gene and reducing the content of intracellular ATP. The data are expressed as the mean ± SD of independent experiments, and the differences in ROS, ATP content or MLKL gene expression are evaluated by Tukey's test statistical analysis. Different statistical letters a, b and c in the figure indicate statistical differences (P<0.05).

In this way, the present invention explores carnosine nanoparticles with different concentrations of 0.5, 1, 5, 10, and 15 mM. The effect of sub-inhibition on the mechanism of action of human colon cancer HCT-116 cell line. The experimental results showed that the viability of HCT-116 cells was significantly decreased after treatment with 5, 10 or 15 mM carnosine nanoparticles for 72 and 96 hours. The HCT-116 cell line was further treated with 0.5, 1, 5, 10 or 15 mM carnosine nanoparticles for 96 hours for follow-up experiments, and it was found that carnosine nanoparticles could increase the expression of Bax, Caspase 8, 3, 9 and PARP genes in cancer cells Promote apoptosis of cancer cells; increase the expression of PI3K III, Beclin 1 and LC 3 genes to promote autophagy in cancer cells; increase the content of ROS and MLKL gene expression in cancer cells, and reduce ATP content to promote cancer cell necrosis/programmed necrosis. Based on the above results, it can be seen that carnosine nanoparticles can inhibit the growth of cancer cells by regulating the apoptosis, autophagy, and necrosis/programmed necrosis mechanism of HCT-116 cell line, thereby inhibiting cancer.

To sum up, the present invention relates to a carnosine nanoparticle used to prepare and regulate colorectal cancer cells. The use of drugs can effectively improve various shortcomings of conventional methods. The proposed Carnosine nanoparticles (C-NPs) can regulate HCT-116 cell line apoptosis (Apoptosis), cell autophagy (Autophagy) and cellular Necrosis/Necroptosis mechanism inhibits the growth of cancer cells to inhibit the effect of cancer, thereby making the invention more advanced, more practical, and more in line with the needs of users, which has indeed met the requirements of the invention patent application , to file a patent application in accordance with the law.

However, the above are only preferred embodiments of the present invention, and should not be limited to this The scope of implementation of the present invention; therefore, all simple equivalent changes and modifications made according to the scope of the patent application of the present invention and the contents of the description of the invention should still fall within the scope of the patent of the present invention.

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Figure 1 is a schematic diagram of the experimental framework for the study of the mechanism of the carnosine nanoparticles of the present invention on inhibiting the human colon cancer HCT-116 cell line. Figure 2A shows the cell morphology of HCT-116 cell line treated with carnosine nanoparticles of the present invention for 72 or 96 hours. Figure 2B is a schematic diagram showing the change of the carnosine nanoparticles of the present invention on the cell viability of the HCT-116 cell line. Figure 3 is a schematic diagram showing the changes of the carnosine nanoparticles of the present invention on the apoptosis of HCT-116 cell line. Figure 4A is a schematic diagram showing the change of Bax gene expression in HCT-116 cell line by carnosine nanoparticles of the present invention. Figure 4B is a schematic diagram showing the change in the expression of the Bcl 2 gene of HCT-116 cell line by the carnosine nanoparticles of the present invention. Figure 4C is a schematic diagram of the change in the expression level of the Caspase 9 gene of the HCT-116 cell line by the carnosine nanoparticles of the present invention. Figure 4D is a schematic diagram of the change in the expression of the Caspase 3 gene of the HCT-116 cell line by the carnosine nanoparticles of the present invention. Figure 4E is a schematic diagram showing the change in the expression of the Caspase 8 gene of the HCT-116 cell line by the carnosine nanoparticles of the present invention. Figure 4F is a schematic diagram showing the change in the expression level of the PARP gene of the HCT-116 cell line by the carnosine nanoparticles of the present invention. Fig. 5 is a schematic diagram showing the change of autophagy of HCT-116 cell line by carnosine nanoparticles of the present invention. Figure 6A is a schematic diagram showing the change of the carnosine nanoparticles of the present invention on the expression of Beclin 1 gene in HCT-116 cell line. Figure 6B is a schematic diagram of the change in the expression level of the PI3K III gene of the HCT-116 cell line by the carnosine nanoparticles of the present invention. Figure 6C is a schematic diagram of the change in the expression of the LC 3 gene of the HCT-116 cell line by the carnosine nanoparticles of the present invention. Figure 7A is a schematic diagram showing the change of ROS content in HCT-116 cell line by carnosine nanoparticles of the present invention. Figure 7B is a schematic diagram showing the change of ATP content of HCT-116 cell line by carnosine nanoparticles of the present invention. Fig. 7C is a schematic diagram showing the change of MLKL gene expression in HCT-116 cell line by carnosine nanoparticles of the present invention.

Claims (7)

Use of a carnosine nanoparticle for preparing a drug for regulating intestinal cancer cells, comprising administering an effective dose of a compound or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier; the compound is a carnosine nanoparticle ( Carnosine nanoparticles, C-NPs) with a molecular weight of 453.2 and a density of 1.019, its structural formula is as follows:

; wherein, the carnosine nanoparticle is administered at a dose concentration of 0.5 to 15 mM, and the colorectal cancer cells are human colon cancer HCT-116 cell lines. According to the use of the carnosine nanoparticle described in the first item of the application scope for preparing a drug for regulating intestinal cancer cells, the drug is used for regulating HCT-116 cell line apoptosis (Apoptosis), cell autophagy ( Autophagy) and cell necrosis/necroptosis (Necrosis/Necroptosis) mechanism inhibits the growth of cancer cells and inhibits the formation of cancer. According to the use of the carnosine nanoparticle described in item 1 of the application scope for preparing a drug for regulating intestinal cancer cells, the drug is mixed with a culture medium to treat HCT-116 cell line. According to the use of the carnosine nanoparticles described in item 1 of the claimed scope for preparing a drug for regulating intestinal cancer cells, the carrier comprises excipients, diluents, thickeners, fillers, Binder, disintegrant, lubricant, oily or non-oily base, surfactant, suspending agent, gelling agents, adjuvants, preservatives, antioxidants, stabilizers, colors, or flavors. According to the use of the carnosine nanoparticle described in item 1 of the scope of application for preparing a medicine for regulating intestinal cancer cells, the dosage form of the medicine is a solution. Use of the carnosine nanoparticle described in item 1 of the application scope for preparing a drug for regulating intestinal cancer cells, wherein the carnosine used in the carnosine nanoparticle is (2S)-2-[(3 -Amino-1-oxopropyl)amino]-3-(3H-imidazol-4-yl)propanoic acid with a molecular weight of 226.23 and a density of 1.4. The carnosine nanoparticles described in the first item of the patent scope of the application are used to prepare the regulation of intestinal Use of a drug for cancer cells, wherein the drug is administered to the carnosine nanoparticle for 72 or 96 hours processing time.

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