TWI648051B - Use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells - Google Patents

Abstract

The use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, and the culture of human colon cancer (HCT-116) cell line as an experimental model to investigate the effect of carnosine on the metastasis of human colon cancer cell lines. From the results, carnosine can reduce the metastatic ability of colon cancer cells by regulating cell migration, invasion, intravasation, extravasation, and adhesion.

Description

Use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells

The present invention relates to the use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, in particular to a carnosine which can regulate cell migration, invasion, intravasation, and external Extravasation and adhesion to reduce the metastatic ability of colon cancer (HCT-116) cells.

With the development of social economy, the lifestyle of Chinese people has changed, and the incidence of cancer has increased year by year due to the influence of dietary refinement and westernization. According to the International Agency for Research on Cancer (IARC) report, about 1.2 million people worldwide suffered from colon cancer in 2012 (IARC, 2016); the World Health Organization (WHO) also pointed out In 2015, 694,000 people died of colon cancer (WHO, 2016). In addition, among the top ten causes of death in Taiwan announced by the Ministry of Health and Welfare of the Republic of China in 2017, malignant tumors once again ranked first among the top ten causes of death among Chinese people. Moreover, among the top ten causes of cancer, colon cancer is not only high. The number of deaths due to colon cancer is as high as 24.3 per 100,000 population (Ministry of Health and Welfare, 2017). It can be seen that colon cancer poses a major threat to the health of our people. Therefore, how to prevent the occurrence of colon cancer has become one of the important topics for many scholars to study.

It is known that metastasis of cancer cells is one of the main causes of poor treatment of cancer. Once the cancer metastasizes, the prognosis is quite poor, and more than 90% of cancer patients die because cancer cells are transferred to Caused by distal organs (Leber and Efferth, 2009). It is worth noting that in the advanced stage of colon cancer, cancer cells often The distant metastasis from the lymph or blood to the liver, lungs, bones and brain (Roth et al., 2009) poses a hazard to human health.

At present, there are surgical resection, chemotherapy, target drugs combined with radiotherapy for clinical treatment of colon cancer, but these treatments often have different side effects (Li et al., 2010). Therefore, in recent years, many natural active ingredients that have the effect of preventing and improving the treatment of cancer have received increasing attention. How to apply natural foods and their active ingredients to reduce the metastasis of cancer cells has become an important research topic.

Carnosine is an endogenous double peptide existing in skeletal muscle of vertebrate. It is known to have anti-fatigue, anti-oxidation, high blood pressure, diabetes and inhibition of cancer cell growth. However, the effects of carnosine on human colorectal cancer cell metastasis have been rarely studied.

In view of the fact that the regulation mechanism of carnosine inhibiting cancer cell metastasis is still incomplete, the general practitioners cannot meet the needs of users in actual use.

The main object of the present invention is to overcome the above problems encountered in the prior art and to provide a carnosine which can regulate cell migration, invasion, intravasation, extravasation by extravasation. And the use of a compound to reduce the metastatic ability of colon cancer (HCT-116) cells for the preparation of a medicament for regulating metastasis of intestinal cancer cells.

For the purposes of the above, the present invention is a use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, comprising an effective amount of a compound of the formula I - carnosine (2S)-2-[(3-Amino-1-oxopropyl) Amino]-3-(3H-imidazol-4-yl)propanoic acid,carnosine)

Or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In the above embodiments of the invention, the compound treats cancer.

In the above embodiments of the invention, the cancer comprises colon cancer.

In the above embodiment of the invention, the intestinal cancer cell is human colon cancer cell line HCT-116.

In the above examples of the invention, the concentration of the compound of formula I - carnosine is from 0.5 mM to 5 mM.

Fig. 1 is a schematic diagram showing the regulation process of carnosine of the present invention for human colon cancer cell metastasis.

Fig. 2 is a graph showing the effect of carnosine of the present invention on cell viability of HCT-116 cell line.

Fig. 3 is a graph showing the effect of the carnosine of the present invention on the migration ability of the HCT-116 cell line.

Fig. 4 is a graph showing the effect of the carnosine of the present invention on the invasive ability of the HCT-116 cell line.

Fig. 5 is a schematic view showing the effect of the carnosine of the present invention on the MMP-2 gene expression of the HCT-116 cell line.

Fig. 6 is a graph showing the effect of the carnosine of the present invention on the MMP-9 gene expression of the HCT-116 cell line.

Figure 7 is a graph showing the effect of carnosine of the present invention on TEER of EA.hy 926 cell line.

Fig. 8 is a schematic view showing the effect of carnosine of the present invention on adhesion of HCT-116 cell line to EA.hy 926 cell line.

Fig. 9 is a schematic view showing the therapeutic effect of carnosine after expression of the transfer regulatory protein of the present invention.

Fig. 10 is a photomicrograph showing the effect of the carnosine of the present invention on the cell type of the HCT-116 cell line.

Please refer to FIG. 1 to FIG. 10 , which are schematic diagrams showing the regulation process of carnosine-to-human colon cancer cell metastasis of the present invention, and the effect of carnosine of the present invention on cell viability of HCT-116 cell line, The effect of the carnosine of the present invention on the migration ability of the HCT-116 cell line, the effect of the carnosine of the present invention on the invasion ability of the HCT-116 cell line, and the MMP-2 gene expression of the carnosine of the present invention on the HCT-116 cell line Schematic diagram of the influence of the long strip, the effect of the carnosine of the present invention on the MMP-9 gene expression of the HCT-116 cell line, the effect of the carnosine of the present invention on the TEER of the EA.hy 926 cell line, and the carnosine pair HCT- of the present invention Schematic diagram of the effect of the 116 cell line adhering to the EA.hy 926 cell line, the therapeutic effect of the carnosine after the expression of the transfer regulatory protein of the present invention, and the micrograph of the effect of the carnosine of the present invention on the cell type of the HCT-116 cell line. As shown in the figure: The present invention is a use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, using carnosine as an experimental material, and using human colon cancer HCT-116 cell strain to culture different concentrations of carnosine. After treatment for different time, the effects of carnosine on cell viability were investigated by MTT assay and morphology examination. The ability of carnosine to migrate to cancer cells was explored in conjunction with the wound healing assay and the analysis of E-cadherin; cell invasion assay and matrix metalloproteinases (MMPs) were used. MMP-2 and MMP-9 performance, explore carnosine to cancer The impact of cell invasion. Fluorescently labeled HCT-116 cells were co-cultured with human vascular endothelium (EA.hy 926) cells for cell adhesion assay and analysis of integrin β1 and E-selectin (E- Selectin) and cell adhesion molecule-1 (ICAM-1) showed the effect of carnosine on the ability of cancer cells to adhere to endothelial cells. The endothelial cell permeability model was used to analyze endothelial cell permeability and phosphorylation of vascular endothelial cadherin (VE-cadherin). As shown in the experimental scheme of Figure 1, it is expected that the present invention can be understood to understand the migration, invasion, invasion, extravasation, extravasation and adhesion of carnosine to human colon cancer HCT-116 cell line. The role of (adhesion).

The promoter region of the transcription factor NF-κB is contained in the promoter regions of E-cadherin, MMP-2, MMP-9, Integrin β1, E-selectin, ICAM-1 and VE-cadherin. Therefore, the experimental model will also explore whether carnosine affects cancer cell metastasis via regulation of NF-κB activation.

The following examples are merely illustrative of the invention, but the scope of protection of the invention is not limited to the following examples. The above and other objects, features and advantages of the present invention will become more apparent and understood.

[Example 1] Experimental materials

Carnosine source

The compound used in this experiment, carnosine (2S)-2-[(3-Amino-1-oxopropyl)amino]-3-(3H-imidazol-4-yl)propanoic acid, carnosine), was purchased at Sigma ( Sigma-Aldrich Co. USA).

Cell culture

Human colon cancer HCT-116 cell line

The cell line used in this experiment was human colon cancer HCT-116 cell line, which was purchased from the Center for Biological Resource Conservation and Research of the Food Industry Development Research Institute. The HCT-116 cell line was cultured in McCoy's 5A medium containing 2.2 g/L sodium bicarbonate, 1% penicillin/streptomycin 100 U/mL and 10% fetal bovine serum. The medium was cultured in a constant temperature incubator containing 5% carbon dioxide (CO ₂ ) at 37 °C.

The cell cryotube was taken out in a liquid barrel and completely dissolved in a 37 ° C water bath. After disinfection, the thawed cell liquid was taken out in a sterile tube and placed in a centrifuge tube containing McCoy's 5A medium, centrifuged at 1000 rpm for 2 minutes, and removed. After clearing the solution, add fresh McCoy's 5A medium and mix the cells thoroughly. Then, mix the cell liquid into a 10 cm culture dish and shake it gently to make the cells evenly dispersed. Finally, place the culture dish at 37 ° C with 5% CO _{2 .} Cultivate in the incubator. On the next day, it was determined whether the cells were attached to the culture dish and replaced with fresh McCoy's 5A medium for culture.

The cells were grown in petri dishes until they were nine minutes full, and consisted of 9 Mm disodium hydrogen phosphate (Na ₂ HPO ₄ ), 140 mM sodium chloride (NaCl), and 1 mM sodium dihydrogen phosphate (NaH ₂ PO ₄ ) to form pH 7.4 phosphoric acid. After washing twice with phosphate buffered saline (PBS), add 0.25% trypsin (trypsin/EDTA) and place in a 37 ° C incubator with 5% CO ₂ for 3 minutes, then take the plate and wash it down with McCoy's 5A medium. The cells were collected, and the cell liquid was collected into a centrifuge tube, centrifuged at 1000 rpm for 2 minutes, the supernatant was removed, the cells were thoroughly mixed by adding fresh McCoy's 5A medium, and the desired cell liquid was taken out into a 10 cm culture dish and left and right. The cells were evenly dispersed by gentle shaking, and finally the dishes were incubated in an incubator containing 5% CO ₂ at 37 °C. On the next day, it was determined whether the cells were attached to the culture dish and replaced with fresh McCoy's 5A medium for culture.

Count the number of cells to 1 × 10 ⁶ cells per ml using a hemocytometer, add 1 mL of McCoy's 5A medium containing 10% dimethyl sulfoxide (DMSO), and mix the cells thoroughly, then transfer to a cryotube and place. The library was frozen at -80 ° C and then transferred to a liquid nitrogen drum for 24 hours.

2. Human vascular endothelial EA.hy 926 cell line

The human vascular endothelial EA.hy 926 cell strain used in this experiment was provided by the laboratory of Teacher Li Zonggui of China Medical University. The EA.hy 926 cell line is cultured to contain 3.7 g/L sodium bicarbonate, 1% Non-Essential Amino Acid (NEAA), 1% sodium pyruvate, 1% penicillin/streptococcus In a DMEM (Dulbecco's Modified Eagle Medium) medium containing 100 U/mL and 10% fetal calf serum, it was cultured in an incubator containing 5% CO ₂ at 37 °C.

The cell cryotube was taken out in a liquid barrel and immediately placed in a 37 ° C water bath to completely dissolve it. After disinfection, the thawed cell liquid was taken out in a sterile tube and placed in a centrifuge tube containing DMEM medium, and centrifuged at 1000 rpm for 2 minutes in a centrifuge. After removing the supernatant, add fresh DMEM medium and mix the cells thoroughly. Then, mix the cell solution into a 10 cm culture dish and shake it gently to make the cells evenly dispersed. Finally, the culture dish is placed at 37 ° C and contains 5%. Culture in a CO ₂ incubator. On the next day, it was determined whether the cells were attached to the culture dish and replaced with fresh DMEM medium for culture.

When the cells were grown in a petri dish until the end of nine minutes, the cells were washed twice with PBS, and trypsin was added to an incubator containing 5% CO ₂ at 37 ° C for 3 minutes, and the cells were plated, and the cells were washed down with DMEM medium. The cell fluid was collected into a centrifuge tube, centrifuged at 1000 rpm for 2 minutes, the supernatant was removed, fresh DMEM medium was added and the cells were thoroughly mixed, and the desired cell solution was taken out into a 10 cm culture dish and gently shaken left and right. The cells were uniformly dispersed, and finally the dishes were incubated in an incubator containing 5% CO ₂ at 37 °C. On the next day, it was determined whether the cells were attached to the culture dish and replaced with fresh DMEM medium for culture.

Count the number of cells to 5 × 10 ⁵ cells per ml using a hemocytometer, add 1 mL of DMEM medium containing 10% DMSO and mix the cells well, transfer to a cryotube, and place in a freezer at -80 ° C, after 24 hours. Move to a liquid nitrogen drum for cryopreservation.

[Example 2] Experimental method

Sample processing

The HCT-116 cell line and the EA.hy 926 cell line were cultured in a 3.5 cm diameter culture dish at 1 × 10 ⁶ and 5 × 10 ⁵ cells per ml, respectively. After 24 hours of culture, 0.5, 1, or 5 mM carnosine was added, respectively, and after 24 hours of culture, subsequent analysis was performed.

Cell viability

1. MTT assay (MTT assay)

In this experiment, the cell survival rate (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) was used as a method for determining the survival rate of HCT-116 cell line. The succinate dehydrogenase (SDH) and cytochrome C (Cytochrome C) in the mitochondria of living cells will cleave the tetrazolium ring of MTT to form a blue crystalline substance. (formazan), the amount of the crystalline substance produced is proportional to the number of living cells, and the formazan crystal is dissolved by isopropanol, and then determined by an enzyme-linked immuno-sorbent Assay reader (ELISA reader). Absorbance at a wavelength of 570 nm. This absorbance value represents the activity of the granulosa, which is proportional to the number of viable cells, so the MTT assay can be used as an indicator of cell viability.

In this experiment, 1×10 ⁶ HCT-116 cells per ml were planted in a 3.5 cm culture dish, and cultured in a constant temperature incubator containing 5% CO ₂ at 37 ° C until 8 minutes. 0.5, 1, or 5 mM carnosine was administered separately, and the medium containing only sterile water was used as the control group. After 24 and 48 hours of culture, the medium was removed, washed twice with PBS, and added to a medium containing 0.5 mg/mL MTT, and cultured in a constant temperature incubator at 37 ° C and 5% CO ₂ for 3 hours to remove the culture solution and added. 1 mL of isopropanol was dissolved in formazan, shaken for 10 minutes, transferred to a microcentrifuge tube, centrifuged at 7,500 rpm for 5 minutes, and 200 μL of the supernatant was taken in a 96-well plate. The absorbance at 570 nm was measured by ELISA reader. .

2. Cell morphology observation and record

In this experiment, 1×10 ⁶ HCT-116 cells per ml were planted in a 3.5 cm culture dish, and cultured in a constant temperature incubator containing 5% CO ₂ at 37 ° C until 8 minutes. 0.5, 1, or 5 mM carnosine was administered separately, and the medium containing only sterile water was used as the control group. After 24 and 48 hours of culture, the number and morphology of the cells were observed and recorded by an inverted phase contrast microscope.

Analysis of wound healing ability of carnosine on HCT-116 cell line

In this experiment, the experimental method of Ang et al. (2010) was used to make the cell culture plate produce a damaged area, and the cell movement process was used to change the damage area, thereby indicating the ability of cell migration.

In this experiment, 1×10 ⁶ HCT-116 cells per ml were cultured in a 3.5 cm culture dish. After the cells were grown to full volume, the culture dish was lightly drawn with a 10 μL sterile micropipette tip to create a damaged area, and then washed with PBS. After treatment with 0.5, 1, or 5 mM carnosine for 0, 12, 24, and 48 hours, the cell migration was observed and recorded using an inverted phase contrast microscope; the damage area was further examined using Image J software (Image J 1.47t) , National Institutes of Health, USA).

Analysis of carnosine on the expression of metastasis-associated genes in HCT-116 cell line

1. Preparation of carnosine pair

In this experiment, 1×10 ⁶ HCT-116 cells per ml were planted in a 3.5 cm culture dish, and cultured in a constant temperature incubator containing 5% CO ₂ at 37 ° C until 8 minutes. 0.5, 1, or 5 mM carnosine was administered separately, and cultured in a medium containing only sterile water for 24 hours.

2. Extraction of total RNA from HCT-116 cell line

Prior to the experiment, all items must be autoclaved to remove residual RNase. Take the above cells, wash twice with cold PBS, add 1 mL of nucleic acid protein three-phase extraction reagent (TRI reagent) and mix well with shaking, add 200 μL chloroform, gently shake and mix, ice bath for 5 minutes, at 4 ° C, Centrifuge at 13000 rpm for 15 minutes, take the supernatant into a new tube, add an equal volume of isopropanol (ie 1:1), shake well and centrifuge, then ice bath for 5 minutes, centrifuge at 13000 rpm for 15 minutes at 4 ° C, aspirate Clear the supernatant, leave the bottom RNA, add 7% alcohol 1m, centrifuge at 4 ° C, 13000 rpm for 10 minutes, aspirate the supernatant, leave a precipitate, and then detach at 4 ° C, 13000 rpm for 1 minute, remove the residual alcohol After that, the RNA pellet was naturally air-dried for 15 minutes, finally dissolved in DEPC-H ₂ O solution, quantified and stored at -20 ° C until use.

3. Quantification of total RNA

The total RNA was determined by the nucleic acid protein analysis system. Before the measurement, 2 μL of DEPC-H ₂ O (as a control) was taken in the sample placement tank. After the measurement was completed, it was placed in the sample placement tank and wiped with DEPC-H ₂ O. Then, 2 μL of the whole amount of ribonucleic acid solution is added to the sample placement tank, and after the measurement is completed, the total RNA solution in the sample placement tank is wiped clean, and the above-mentioned action is repeated in sequence to connect the nucleic acid protein quantitative analysis system with the computer. The computer software calculates the sample concentration and the ratio OD260/OD280 after each measurement is completed.

4. Total RNA is reverse transcribed into complementary DNA (cDNA)

This experiment was carried out in accordance with the method recommended by the Promega manufacturer. The synthesis of single-strand cDNA (first cDNA) was first added to 1 μL of 10 mM deoxynucleotide triphosphate (dNTP), 0.5 μL of oligo (Dt), and 3 μg of RNA in a 1.5 mL microtube. The sterilized water was made up to a total volume of 20 μL, and then the microtubes were placed on a heater at 65 ° C for 5 minutes, and then placed on ice for 5 minutes, followed by the addition of the following reagents: 1 μL of RNasein (RNasin), 1 μL of M- MLV reverse transcriptase (M-MLV Reverse transcriptase), and 5 μL M-MLV RT 5X buffer, the final total volume is 27 μL, all the reagents are evenly mixed, placed in a 37 ° C heating plate for 1 hour, that is, a single strand cDNA solution. Finally placed at -20 ° C for storage.

5. Real-time reverse transcription-polymerase chin reaction (Real-time PCR)

Real-time PCR differs from PCR in that Real-time PCR adds a double-stranded DNA bond to the PCR reaction and generates a fluorescent dye SYBR green I under the excitation of a halogen lamp. While the circulating reaction increases the amount of target gene synthesis, SYBR green I binds to the double-stranded DNA, so the higher the amount of SYBR green I fluorescence, the more the gene expression is. The principle of Real-time PCR is to use different concentrations of the template in the same PCR reaction conditions, the reaction containing high concentration of the template will reach the geometric phase faster, at this time the synthesis of DNA is doubled in the definition, reached in the definition The number of critical PCR cycles at the midpoint of the geometric phase is called the threshold cycle (CT), that is, the CT value increases as the concentration of the template decreases. In order to confirm the operation or not pollution during the whole reaction process, it is necessary to add a negative control group without a template, or a non-template control (NTC), and Each quantification can be compared with each other, and the difference in the efficiency of the PCR reaction can be avoided to lead to quantitative analysis bias. Therefore, more than one internal control (quality control (QC)) is added to each quantitative analysis because of the specificity. The concentration converted by the internal control can be used to confirm the accuracy of each quantification. The actual quantitative reagents were added as follows: 2.5 μL double distilled water (ddH ₂ O), 5 μL 2X SYBR Fast MM, 0.5 μL 20X Target Primer set, and 2 μL cDNA (5 ng), and the final total volume was 10 μL. After the addition of the reaction reagent, the PCR-specific 8-plate was placed in a real-time polymerase chain reaction reactor (LightCyvler 96 Real-Time PCR System, Roche Life Science, Basel, Switzerland) to perform the reaction, and the Real-Time PCR conditions were set as follows. Table 1 shows:

Since the relative quantification requires the constant performance characteristic gene of colon cancer HCT-116 as a necessary calculation value for normal, the test method of the present invention uses colon cancer HCT-116 GAPDH as a control group, and Real-Time PCR is used. Each gene-specific primer is shown in Table 2.

Analysis of the invasive ability of carnosine on HCT-116 cell line

In this experiment, a cell culture mode of a 24-well (transwell) culture dish was used, and 70 μL of a medium containing a matrigel medium (in serum-free McCoy's 5A medium 1) was previously injected into a 8.0 μm cell culture dish. 6 dilution) Incubate in a 37 ° C incubator for 60 minutes. Count 5×10 ⁴ cells per ml, add 200 μL McCoy's 5A medium containing 0.5, 1, or 5 mM carnosine and no FBS to the transwell insert, and add 500 μL of FCo-containing McCoy's 5A medium to the 24-well culture dish. Incubate for 48 hours in an incubator containing 5% CO ₂ at 37 °C. The transwell insert was taken out, washed twice with PBS, and then fixed with 4% formaldehyde solution for 30 minutes, and stained with 0.5% crystal violet for 15 minutes to invade the performance. Finally, 1 mL of methanol was added and shaken at room temperature for 10 minutes to discolor the crystal violet. The methanol was transferred to a microcentrifuge tube and mixed, and 200 μL of the cell solution was taken in a 96-well plate, and its absorbance at a wavelength of 540 nm was measured by an ELISA reader.

[Example 3] Experimental results

Statistical Analysis

The experimental data were statistically analyzed using the analysis of variance (ANOVA) in the SPSS statistical software package with Duncan's test to determine whether there was a significant difference between the experimental data.

1. Effect of cellulite on HCT-116 cell line after 24, 48 and 72 hours of cell viability

HCT-116 cell line was cultured in McCoy's 5A medium containing 0.5, 1 and 5 mM carnosine, and HCT-116 cell line was treated with sterile water as a control group, and cell viability was changed after 24, 48 and 72 hours of treatment. As shown in Figure 2, the data were from four independent experiments, expressed as mean ± SD, and statistically analyzed by Duncan's test to assess significant differences in cell viability, with significant cell viability between the different letters in the figure. Difference (P<0.05).

After treatment with 0.5, 1 and 5 mM carnosine for 24 hours, the viability of HCT-116 cells was 101.3%, 98.9% and 99.63%, respectively, which was significantly lower than that of the control group (100%) in the 1 mM and 5 mM groups. In the control group, after 48 hours, their viability was 96.2%, 91.2% and 86.9%, respectively, which were significantly lower than the control group, and after 72 hours, their viability was 82.5%, 78.9% and 80.2%, respectively. Below the control group (P <0.05). The results show, When the HCT-116 cell line was treated with 1 or 5 mM carnosine for 24, 48 and 72 hours, carnosine has the ability to inhibit the viability of cancer cells.

2. Effect of carnosine on migration ability of HCT-116 cell line

Using inverted microscope, HCT-116 cells were cultured in McCoy's 5A medium containing 0.5, 1 and 5 mM carnosine, and HCT-116 cells were treated with sterile water as control group at treatments 0, 12, 24 and 48. After hours, changes in cancer cell wound healing were shown in Figure 3, with data from four independent experiments, expressed as mean ± SD, and statistical analysis by Duncan's test to assess significant differences in inhibition of cell migration, There was a significant difference in cell migration between the different letters (P < 0.05).

It can be seen from the upper panel of Fig. 3 that the HCT-116 cell line was treated with 0.5, 1 and 5 mM carnosine for 48 hours, and the cell damage area was significantly reduced compared with the control group. It can be seen from the lower panel of Figure 3 that after treatment with 0.5, 1 and 5 mM carnosine for 12 and 24 hours, there was no significant difference between the cancer cell injury area and the control group, but the cell migration ability was 84.9% after 48 hours (control group). ), 79.2%, 74.1%, and 68.8%, all significantly lower than the control group (P <0.05). From the above results, it was found that when the HCT-116 cell line was treated with 0.5, 1 or 5 mM carnosine for 48 hours, the migration ability of the HCT-116 cell line was significantly inhibited.

3. Effect of carnosine on the invasion ability of HCT-116 cell line

Figure 4 shows that HCT-116 cells were cultured in McCoy's 5A medium containing 0.5, 1, and 5 mM carnosine, and HCT-116 cells were treated with sterile water as control group. After 24 hours of electroplating, cells were filtered. The top of the device is removed, the cells invading the membrane are fixed and counted, and the invasive ability is changed. The data is from four independent experiments, with an average value. ±SD indicates that statistical analysis of Duncan’s test was performed to assess significant differences in cell invasion, and there was a significant difference in cell invasion between the different letters in the figure (P<0.05).

It can be seen from the above figure in Fig. 4 that the HCT-116 cell line treated with 0.5, 1 and 5 mM carnosine for 24 hours, the higher the concentration of carnosine in the purple-blue block, the less the color, and the difference compared with the control group. It shows that the invasion ability of HCT-116 cell line is decreased. From the lower panel of Fig. 4, the invasive ability after treatment with 0.5, 1 and 5 mM carnosine for 24 hours was 94.3%, 65.5% and 52.3%, respectively, which was significantly lower than that of the control group (P<0.05). From the above results, it was found that HCT-116 cell line treated with 1 or 5 mM carnosine for 24 hours significantly inhibited the invasion of HCT-116 and significantly increased the expression of E-cadherin protein (P<0.05).

4. Effect of carnosine on the expression of MMP-2 gene in HCT-116 cell line

Figure 5 shows that HCT-116 cells were cultured in McCoy's 5A medium containing 0.5, 1 and 5 mM carnosine, and HCT-116 cells were treated with sterile water as control group. After treatment for 24 hours, MMP-2 was treated. The effects of gene expression, including data from four independent experiments, expressed as mean ± SD, and statistical analysis by Duncan's test to assess significant differences in MMP-2 gene expression, showing MMP-2 gene expression between different letters in the figure There was a significant difference (P < 0.05).

The HMP-116 cell line was treated with 0.5, 1 and 5 mM carnosine for 24 hours, and the MMP-2 genes were 5.2, 9.6 and 8.4, respectively, which were significantly higher than those of the control group (P<0.05).

5. Effect of carnosine on the expression of MMP-9 gene in HCT-116 cell line

Figure 6 shows that HCT-116 cells were cultured in McCoy's 5A medium containing 0.5, 1 and 5 mM carnosine, and HCT-116 cells were treated with sterile water as control group. After 24 hours, the effect of MMP-9 gene expression, data from four independent experiments, expressed as mean ± SD, and statistical analysis by Duncan's test to assess the significant difference in MMP-9 gene performance, different in the figure There was a significant difference in the expression of MMP-9 gene between the letters (P<0.05).

The HMP-116 cells were treated with carnosine at 0.5 and 1 mM for 24 hours, respectively, and the MMP-9 genes were 0.9 and 0.7, respectively, which were significantly lower than the control group (P < 0.05).

6. Effect of carnosine on trans epithelial electric resistance (TEER) of EA.hy 926 cell line

Figure 7 shows the effect of EA.hy 926 cell line treated with 0.5, 1 and 5 mM carnosine and treated with sterile water as EA.hy 926 cell line as control group for 24 hours after treatment. The TEER of the EA.hy 926 cell line treated with carnosine at 0.5, 1 and 5 mM for 24 hours was significantly lower than that of the control group (P < 0.05).

7. Effect of carnosine on adhesion of HCT-116 cell line to EA.hy 926 cell line

From the results of cell adhesion analysis shown in Fig. 8, it can be seen that the treatment of EA.hy 926 cell line with 0.5, 1 or 5 mM carnosine for 24 hours significantly reduced the adhesion of HCT-116 to EA.hy926 cell line (P<0.05). ). Figure 9 shows the therapeutic effect of carnosine after migration of regulatory proteins. After treatment with HCT-116 cells with 1 or 5 mM carnosine, the expression of Integrin β1 in HCT-116 cells was significantly inhibited (P<0.05). The expression of E-selectin and ICAM-1 in EA.hy926 endothelial cells was decreased (P<0.05).

8. Effect of carnosine on HTD-116 cell line after cell morphology

Observation by electron microscopy showed that HCT-116 cells were treated with 0.5, 1 and 5 mM carnosine, and HCT-116 cells were treated with sterile water as control group. After treatment for different time, the cell morphology changed as shown in Fig. 10. Shown. The HCT-116 cell line was treated with different doses for different time, and its cell morphology was observed by electron microscopy. The decrease in cell number was significantly different from that of the control group. From the above results, it was found that when the HCT-116 cell line was treated with 1 or 5 mM carnosine for various times, carnosine has the ability to inhibit the proliferation of cancer cells.

Therefore, the present invention uses human colon cancer (HCT-116) cell line culture as an experimental model to investigate the effect of carnosine on the metastasis of human colon cancer cell lines. It was found that when the HCT-116 cells were treated with 0.5, 1 or 5 mM carnosine for 48 hours, the migration ability of HCT-116 cells was significantly inhibited (P<0.05); treatment with 0.5, 1 or 5 mM carnosine 48 After an hour, the performance of MMP-9 was significantly reduced (P < 0.05). In addition, treatment of HCT-116 cells with 1 or 5 mM carnosine for 24 hours significantly inhibited the invasion of HCT-116 (P < 0.05) and significantly increased the performance of E-cadherin protein (P < 0.05). From the results of cell adhesion analysis, it was found that treatment of endothelial (EA.hy926) cell line with 0.5, 1 or 5 mM carnosine significantly reduced the adhesion of HCT-116 to EA.hy926 cell line (P<0.05). Treatment of HCT-116 cell line with 1 or 5 mM carnosine significantly inhibited the expression of Integrin β1 in HCT-116 cell line (P<0.05), and significantly decreased the adhesion factor E-selectin and ICAM- in EA.hy926 endothelial cells. 1 performance (P <0.05). Observed by scanning electron microscopy, carnosine can reduce LPS-induced cell flattening; increase the transepithelial resistance (TEER) of monolayer cells, and significantly reduce the expression of VE-cadherin phosphorylation in cells (P<0.05). It reduces the permeability of endothelial cells and shows that carnosine has the ability to regulate cell invasion. From the above results, carnosine can reduce the metastatic ability of colon cancer cells by regulating cell migration, invasion, invasion and adhesion.

In summary, the present invention is a use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, which can effectively improve various disadvantages of the conventional use, and the carnosine can regulate cell migration and invasion (invasion). ), intravasation, extravasation, and adhesion to reduce the metastatic ability of colon cancer (HCT-116) cells, thereby making the invention more progressive, more practical, and more user-friendly. It must have met the requirements of the invention patent application and filed a patent application according to law. However, the above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto; therefore, the simple equivalent changes and modifications made in accordance with the scope of the present invention and the contents of the invention are modified. All should remain within the scope of the invention patent.

Claims (4)

Use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, comprising an effective amount of a compound of formula I - carnosine (2S)-2-[(3-Amino-1-oxopropyl)amino]-3-(3H-imidazol -4-yl)propanoic acid, carnosine) Or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; wherein the concentration of the compound of formula I - carnosine is from 0.5 mM to 5 mM. The use according to claim 1, wherein the compound treats cancer. The use according to claim 2, wherein the cancer comprises colon cancer. The use according to claim 1, wherein the intestinal cancer cell is human colon cancer cell line HCT-116.