CN105456247A - Application of nafamostat mesylate in aspect of treating colorectal cancer - Google Patents

Abstract

The invention discloses the application of nafamostat mesylate in the preparation of the medicine for treating intestinal cancer, and the application of the combination of nafamostat mesilate and oxaliplatin in the preparation of the medicine for treating intestinal cancer. The research of the present invention shows that nafamostat mesylate can significantly inhibit the growth and proliferation of colorectal cancer cells, can significantly inhibit the growth and volume increase of colorectal cancer cells, induce the apoptosis of colorectal cancer cells, and inhibit the growth and proliferation of colorectal cancer cells. The metastasis of cancer cells has a significant curative effect on colorectal cancer and can be applied to the prevention and treatment of colorectal cancer. The present invention not only provides a new application of nafamostat mesilate, provides a new field for the application of nafamostat mesilate, but also provides a new therapeutic drug and treatment pathway. Moreover, the present invention also provides a new scheme for treating colorectal cancer with nafamostat mesylate and oxaliplatin in combination, which has a more significant therapeutic effect on colorectal cancer.

Description

Application of nafamostat mesylate in the treatment of colorectal cancer

technical field

The invention belongs to the technical field of medicine. More specifically, it relates to the application of nafamostat mesylate in the treatment of colorectal cancer.

Background technique

Nafamostat mesilate is a protease inhibitor that mainly exerts anti-inflammatory effects by inhibiting tryptase in mast cells. It has been approved by the FDA for the treatment of pancreatitis and disseminated intravascular coagulation. Can be used as an anticoagulant during hemodialysis.

Recent studies have shown that nafamostat mesylate can kill pancreatic cancer cells to a certain extent, and has a certain anti-pancreatic cancer effect in nude mouse animal models.

At present, there are no reports on the application of nafalimus mesylate in other diseases such as other cancers.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the application limitations and shortcomings of the existing nafamostat mesylate, and provide a new application of nafamostat mesylate, that is, the application in the treatment of colorectal cancer.

The object of the present invention is to provide the application of nafamostat mesylate in the preparation of medicine for treating intestinal cancer.

Another object of the present invention is to provide a medicine for treating colorectal cancer.

The above object of the present invention is achieved through the following technical solutions:

Application of nafamostat mesylate in the preparation of medicine for treating intestinal cancer.

Further preferably, the intestinal cancer is colorectal cancer.

Furthermore, the colorectal cancer is stage I to stage IV colorectal cancer; preferably, the colorectal cancer is stage IV colorectal cancer.

In addition, the above application is the application of nafamostat mesylate in the preparation of drugs for inhibiting the proliferation of intestinal cancer cells.

Preferably, the intestinal cancer cells are human colon cancer cells HCT116, human colon cancer cells SW620 and/or human colon adenocarcinoma cells SW1116.

In addition, further, the above-mentioned application is the application of nafamostat mesylate in the preparation of a drug for inhibiting colorectal cancer cell metastasis.

Furthermore, the above-mentioned application is the application of nafamostat mesylate in the preparation of a drug for inducing apoptosis of colorectal cancer cells.

Wherein, the chemical structural formula of the nafamostat mesylate is shown in the following formula (I):

In addition, a medicine for treating intestinal cancer, which comprises nafamostat mesylate.

Preferably, the medicament also includes its pharmaceutically acceptable carrier.

A drug for the prevention of bowel cancer, which contains nafamostat mesylate.

The research of the present invention shows that nafamostat mesylate has a good preventive effect on colorectal cancer, so it can be applied to the prevention and treatment of colorectal cancer.

In addition, the research of the present invention also proves that the combination of nafamostat mesilate and oxaliplatin has a more significant therapeutic effect on colorectal cancer, so the combination of nafamostat mesilate and oxaliplatin The application in the preparation of medicines for treating intestinal cancer is also within the protection scope of the present invention.

A medicine for preventing and/or treating intestinal cancer, which comprises nafamostat mesylate and oxaliplatin.

Preferably, the medicament also includes its pharmaceutically acceptable carrier.

The present invention has the following beneficial effects:

The present invention provides a new application of nafamostat mesylate, which is applied to the treatment of colorectal cancer. Nafamostat mesylate can significantly inhibit the growth and proliferation of colorectal cancer cells, and can significantly inhibit colorectal cancer. The growth and volume of cancer can be increased, the apoptosis of colorectal cancer cells can be induced, and the metastasis of colorectal cancer cells can be inhibited.

The invention not only provides a new therapeutic drug and a therapeutic approach for the treatment of colorectal cancer, but also provides a new field for the application of nafamostat mesylate.

In addition, the present invention also provides a new scheme for treating colorectal cancer with nafamostat mesylate and oxaliplatin in combination, which has a more significant therapeutic effect on colorectal cancer.

Description of drawings

Figure 1 shows the percentage of surviving cells after treatment of colorectal cancer cells with nafamostat mesylate.

Figure 2 is the effect of nafamostat mesylate on tumor formation in nude mice.

Fig. 3 is the results of inhibition of phosphorylation of IκBα by nafamostat mesylate.

Figure 4 is the result of inhibition of Erk phosphorylation by nafamostat mesylate.

Figure 5 shows the effect of nafamostat mesylate on the proliferation of colorectal cancer cells.

Figure 6 shows the effect of nafamostat mesylate on the invasion and metastasis of colorectal cancer cells.

Figure 7 shows the percentage of surviving cells after treatment of colorectal cancer cells with nafamostat mesylate alone or in combination with oxaliplatin.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

The influence of embodiment 1 nafamostat mesylate on colon cancer cells and tumor formation

1. Experimental materials

(1) Drug: nafamostat mesylate.

(2) Colorectal cancer cells: human colon cancer cells HCT116 and human colon adenocarcinoma cells SW1116.

(3) Apoptosis kit.

(4) Female nude mice, 4-5 weeks old.

2. Experimental grouping

(1) Control group: blank control, that is, colorectal cancer cells were not treated with any drugs.

(2) Experimental group: colorectal cancer cells were treated with nafamostat mesylate.

3. The effect of nafamostat mesylate on the apoptosis of colorectal cancer cells detected by flow cytometry

(1) We paved colorectal cancer cells in six-well plates, and after they adhered to the wall, added 200 μM nafamostat mesylate to treat the cells for 48 hours, and then detected cell apoptosis by flow cytometry.

The specific method is as follows:

1) After the HCT116 and SW1116 cells in the logarithmic growth phase were digested with trypsin, 4×10 ⁵ cells were spread evenly in a six-well plate.

2) After 12 hours, the cells adhered to the wall, and the medium containing 200 μM nafamostat mesylate was replaced at different time points.

3) After the cells were digested, the cells were processed according to the method of the apoptosis detection kit, and the apoptosis of the cells was detected by flow cytometry after AnnexinV and PI staining.

(2) Experimental results

The results are shown in Figure 1. The proportion of AnnexinV and PI double-negative (ie surviving cells) shown in Figure 1, after treatment with nafamostat mesylate, more than half of the colorectal cancer cells died, or even almost completely killed die. It shows that nafamostat mesylate can induce the apoptosis of colorectal cancer cells.

4. Effect of nafamostat mesylate on tumor formation

(1) In the in vivo animal model, we subcutaneously injected intestinal cancer cells (1×10 ⁶ /mouse) into nude mice. After the tumor volume reached 100 mm ³ , we gave nafamostat mesylate (30 mg/kg, three times a week) ) intraperitoneal injection, and observe the changes of the tumor.

The specific method is as follows:

1) Colon cancer HCT116 cells in the logarithmic growth phase were cultured, digested with trypsin, counted to a concentration of 1×10 ⁷ /ml, resuspended in PBS, and transported to the animal laboratory by ice box.

2) Nude mice aged 4-5 weeks were selected and randomly divided into two groups, and each nude mouse was subcutaneously inoculated with 100 μl of the above cell suspension.

3) Observe the tumor formation in nude mice, and measure its longest diameter (L) and shortest diameter (S). After the tumor volume (V=L×S ² /2) reaches 100mm ³ , start to give treatment group nude mice Nafamostat sulfonate (30mg/kg, three times a week) was injected intraperitoneally.

4) The tumor volume of nude mice was measured every three days, and the animals were sacrificed after 3 weeks, and the tumors were taken out and photographed.

(2) Experimental results

The results are shown in Figure 2. The tumor volume of the treatment group (n=8) was significantly smaller than that of the control group, indicating that nafamostat mesylate can significantly inhibit the growth and volume increase of colorectal cancer, and has a positive effect on colorectal cancer. Significant curative effect.

Example 2 Effect of nafamostat mesylate on expression of colorectal cancer cell signaling pathway proteins

1. Experimental materials

(1) Drug: nafamostat mesylate.

(2) Colorectal cancer cells: human colon cancer cells HCT116, SW620 and human colon adenocarcinoma cells SW1116.

(3) IκBα, p-IκBα, Erk, p-Erk and GAPDH antibodies.

2. Western blotting method to detect the effect of nafamostat mesylate on the expression of signaling pathway proteins in colorectal cancer cells

(1) We paved the colorectal cancer cells in a six-well plate, and after they adhered to the wall, we added 50 μM and 100 μM nafamostat mesylate to treat the cells for 24 hours, and then detected nafamostat mesylate by immunoblotting His expression of signaling pathway proteins in colorectal cancer cells.

The specific method is as follows:

1) After HCT116, SW1116 and SW620 cells in the logarithmic growth phase were digested with trypsin, 8×10 ⁵ cells were spread evenly in a six-well plate.

2) After 12 hours, the cells adhered to the wall, and the medium containing different concentrations of nafamostat mesylate was replaced.

3) After adding the drug for 24 hours, collect the cells, use RIPA lysate to obtain the cell protein, quantify it by BCA, take 40 μg protein for SDS-PAGE electrophoresis experiment, and incubate with the corresponding primary antibody after transfer to the membrane, overnight at 4°C.

4) Wash the membrane with TBST, incubate the secondary antibody at room temperature for 2 hours, wash the membrane, and detect the expression of the corresponding protein by chemiluminescence

(2) Experimental results

The results are shown in Figures 3 and 4. After the colorectal cancer cells were treated with nafamostat mesylate, the phosphorylation of IκBα and Erk, which play an important role in the development of tumors, was significantly inhibited, indicating that the corresponding NF-κB and Erk signaling pathway is involved in the anti-colorectal cancer effect of nafamostat mesylate.

Example 3 Effect of nafamostat mesylate on the proliferation of colorectal cancer cells

1. Experimental materials

(1) Drug: nafamostat mesylate.

(2) Colorectal cancer cells: human colon cancer cells HCT116 and human colon adenocarcinoma cells SW1116.

(3) CCK-8 kit.

2. Detect the viability of HCT116 and SW1116 cells after nafamostat mesylate treatment by CCK-8 method

(1) We spread colorectal cancer cells in a 96-well plate, and after they adhered to the wall, we added 0 μM, 200 μM, and 300 μM nafamostat mesylate to treat the cells, and then treated the cells at 6h, 12h, 24h, 36h, and 48h. The cell viability of HCT116 and SW1116 was detected by CCK-8 method.

The specific method is as follows:

1) After the HCT116, SW1116 and SW620 cells in the logarithmic growth phase were digested with trypsin, 4×10 ³ cells were spread evenly in a 96-well plate.

2) After 12 hours, the cells adhered to the wall, and the medium containing different concentrations of nafamostat mesylate was replaced.

3) After adding the drug for 6h, 12h, 24h, 36h, and 48h, add CCK-8 reagent at a ratio of 1:10, incubate at 37°C for 2h, and use a microplate reader to detect the absorbance at 450nm.

(2) Experimental results

The results are shown in Figure 5. After the colorectal cancer cells were treated with nafamostat mesylate, the cell viability decreased significantly, indicating that nafamostat mesylate could significantly inhibit the proliferation of colorectal cancer cells, and the inhibitory effect was time-dependent. and concentration dependence.

Example 4 Effect of Nafamostat Mesylate on the Metastasis of Colorectal Cancer Cells

1. Experimental materials

(1) Drug: nafamostat mesylate.

(2) Colorectal cancer cells: human colon cancer cells HCT116 and human colon adenocarcinoma cells SW1116.

(3) Transwell chambers with and without pre-coated Matrigel.

2. The effect of nafamostat mesylate treatment on the invasion and migration of HCT116 and SW1116 cells was detected by Transwell chamber

(1) We paved the colorectal cancer cells in a six-well plate, and after they adhered to the wall, we added 100 μM nafamostat mesylate to treat the cells for 0h, 12h and 24h, and then detected nafamostat mesylate by Transwell chamber Effects of His treatment on HCT116 and SW1116 cell invasion and migration.

The specific method is as follows:

1) After the HCT116, SW1116 and SW620 cells in the logarithmic growth phase were digested with trypsin, 6×10 ⁵ cells were spread evenly in a six-well plate.

2) After 12 hours, the cells adhered to the wall, and the medium containing 100 μM nafamostat mesylate was replaced at different time points.

3) After drug treatment, cells were digested with trypsin, washed with PBS, and resuspended in serum-free medium for counting.

4) Take 200 μl of cell suspension, containing 1×10 ⁵ and 2×10 ⁵ cells respectively, and add them to the upper chamber of Transwell without pre-coated Matrigel and coated Matrigel, and add 600 μl of 50% serum to the lower chamber culture medium.

5) Cells were cultured in a 37°C incubator for 36 hours, the small chamber was taken out, fixed with methanol and stained with crystal violet, the remaining cells in the upper chamber were removed with a cotton swab, and then air-dried.

6) Take pictures in an inverted microscope.

(2) Experimental results

The results are shown in Figure 6. After colorectal cancer cells were treated with nafamostat mesylate, the number of cells migrating and invading into the lower chamber decreased significantly, indicating that nafamostat mesylate can significantly inhibit colorectal cancer. Cell migration and invasion, and the inhibitory effect was concentration-dependent.

Embodiment 5 nafamostat mesylate and oxaliplatin combination

1. Experimental materials

(1) Drugs: nafamostat mesylate, oxaliplatin.

(2) Colorectal cancer cells: human colon cancer cells HCT116 and human colon adenocarcinoma cells SW1116.

(3) Apoptosis kit.

2. Experimental grouping

(1) Control group: blank control, that is, colorectal cancer cells were not treated with any drugs.

(2) Single medication group 1: Nafamostat mesylate was used to treat colorectal cancer cells.

(3) Single medication group 2: oxaliplatin was used to treat colorectal cancer cells.

(4) Combined drug group: colorectal cancer cells were treated with nafamostat mesylate and oxaliplatin.

3. Flow cytometry detection of the effects of nafamostat mesylate, oxaliplatin and their combination on the apoptosis of colorectal cancer cells

(1) We paved the colorectal cancer cells in a six-well plate, and after they adhered to the wall, we added 100 μM nafamostat mesylate, 50 μM oxaliplatin, and the combination of the two to treat the cells for 24 hours, and then passed Cell apoptosis was detected by flow cytometry.

The specific method is as follows:

1) After the HCT116 and SW1116 cells in the logarithmic growth phase were digested with trypsin, 4×10 ⁵ cells were spread evenly in a six-well plate.

2) After 12 hours, the cells adhered to the wall, and at different time points, the medium was replaced with a medium containing 100 μM nafamostat mesylate, 50 μM oxaliplatin, or a combination of the two.

3) After the cells were digested, the cells were processed according to the method of the apoptosis detection kit, and the apoptosis of the cells was detected by flow cytometry after AnnexinV and PI staining.

(2) Experimental results

The results are shown in Figure 7. Both nafamostat mesylate or oxaliplatin can kill a certain proportion of colorectal cancer cells, while the proportion of surviving cells in the combined drug group is even less. It shows that the use of nafamostat mesylate combined with oxaliplatin can induce the apoptosis of colorectal cancer cells more significantly than the single drug group, and shows that the combined use of nafamostat mesylate and oxaliplatin has the effect on colorectal cancer cells. The therapeutic effect of cancer has been significantly improved.

Claims (10)

1. the application of Nafamostat Mesilate in the medicine of preparation treatment intestinal cancer.

2. apply according to claim 1, it is characterized in that, described intestinal cancer is colorectal cancer.

3. apply according to claim 2, it is characterized in that, described colorectal cancer is that Ι is to IV phase colorectal cancer.

4. apply according to claim 1, it is characterized in that, described application refers to that Nafamostat Mesilate suppresses the application in the medicine of colorectal cancer cell propagation in preparation.

5. apply according to claim 4, it is characterized in that, described colon-cancer cell is human colon cancer cell HCT116, human colon cancer cell SW620 and/or human colon adenocarcinoma cell SW1116.

6. apply according to claim 4, it is characterized in that, described application refers to that Nafamostat Mesilate suppresses the application in the medicine of colorectal cancer cell transfer in preparation.

7. apply according to claim 4, it is characterized in that, described application refers to the application of Nafamostat Mesilate in the medicine of preparation induction colorectal cancer cell apoptosis.

8. prevent and/or treat a medicine for intestinal cancer, it is characterized in that, this pharmaceutical pack is containing Nafamostat Mesilate.

9. the application of coupling both Nafamostat Mesilate and oxaliplatin in the medicine of preparation treatment intestinal cancer.

10. prevent and/or treat a medicine for intestinal cancer, it is characterized in that, this pharmaceutical pack is containing Nafamostat Mesilate and oxaliplatin.