CN117752665B - Application of ursolic acid as salazosulfapyridine anti-liver cancer sensitizer - Google Patents
Application of ursolic acid as salazosulfapyridine anti-liver cancer sensitizer Download PDFInfo
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- CN117752665B CN117752665B CN202410179207.3A CN202410179207A CN117752665B CN 117752665 B CN117752665 B CN 117752665B CN 202410179207 A CN202410179207 A CN 202410179207A CN 117752665 B CN117752665 B CN 117752665B
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- ursolic acid
- liver cancer
- sulfasalazine
- salazosulfapyridine
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- 229960001940 sulfasalazine Drugs 0.000 title claims abstract description 73
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a new application of ursolic acid in enhancing the anti-liver cancer effect of salazosulfapyridine. The invention discovers that the ursolic acid has the effect of enhancing the iron death of the liver cancer cells induced by the salazosulfapyridine for the first time, has good synergistic effect on the anti-liver cancer treatment of the salazosulfapyridine, and can increase the iron death of the human liver cancer cells induced by the salazosulfapyridine. The human liver cancer cells are treated by adding the low-dose concentration of the ursolic acid and the sulfasalazine, the cell activity is obviously reduced compared with that of a blank group, and the activity of the human liver cancer cells treated by the combination of the ursolic acid and the sulfasalazine is only about 40%; meanwhile, the ursolic acid has good prevention effect on the possible potential hepatotoxic effect of the sulfasalazine, and the combined treatment of the ursolic acid and the sulfasalazine can obviously reduce the level of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase and reduce liver injury from the viewpoint of serum biochemical indexes.
Description
Technical Field
The invention relates to the field of medicines, in particular to a novel application of ursolic acid in enhancing the anti-liver cancer effect of salazosulfapyridine.
Background
Sulfasalazine (Sulfasalazine, SAS) is an iron death inducer, which can target inhibition of cystine/glutamate antiport protein (System Xc-), leading to cysteine starvation, glutathione depletion, and further leading to cell iron death. Sulfasalazine is currently mainly used as a sulfonamide antibacterial agent for the treatment of inflammatory bowel disease and rheumatoid arthritis. The literature reports that sulfasalazine inhibits the growth of certain cancer cells, but long-term large-dose sulfasalazine treatment may have some adverse effects on the gastrointestinal tract, liver and kidneys.
Ursolic Acid (UA) is a pentacyclic triterpene compound that is found in a variety of medicinal plants and fruits and vegetables. Ursolic acid can target tumor suppressor gene, inhibit key signal path of tumor generation, induce endoplasmic reticulum stress, destroy mitochondrial function, inhibit growth, proliferation and metastasis of cancer cells, target different types of programmed cell death, and exert anticancer effect. In addition, when the ursolic acid is used in combination with other cancer therapeutic drugs, the ursolic acid also shows remarkable synergistic effect, and can improve the sensitivity of cancer cells to chemotherapy and radiotherapy. The invention discovers that under a lower dosage, the ursolic acid has a good synergistic effect on the death of liver cancer cell iron induced by low-concentration salazosulfapyridine for the first time, and has a preventive effect on possible hepatotoxicity of salazosulfapyridine.
Patent document CN103070875A discloses a composition with anticancer effect, which mainly comprises the following active ingredients: the composition can effectively improve the effective drug concentration of tumor parts, effectively kill liver cancer cells and reduce the toxic and side effects on normal cells.
Patent document CN109464460a discloses a pharmaceutical composition for improving the sensitivity of anti-tumor drugs and the application thereof in preparing anti-tumor drugs, wherein the composition comprises an effective dose of sulfasalazine and nano zinc oxide, and a pharmaceutically acceptable carrier, and can be used for preparing tumor drug sensitivity agents. The combination of the sulfasalazine and the nano zinc oxide can play a role in synergy, and improve the killing effect of tumor cells of traditional chemotherapeutics, molecular targeting drugs and immunotherapeutic drugs.
However, the prior art has not reported that ursolic acid and sulfasalazine are combined, and has not reported that ursolic acid is used for enhancing the anti-liver cancer efficacy of sulfasalazine. In view of this, the present invention has been made.
Disclosure of Invention
The invention overcomes the defects existing in the prior art and provides a new application of ursolic acid in enhancing the liver cancer resistance of the sulfasalazine.
The present invention provides in a first aspect a pharmaceutical composition comprising ursolic acid and sulfasalazine.
Further, the mass ratio of ursolic acid to sulfasalazine in the pharmaceutical composition is 1-5:20 (such as 1:20, 2:20, 3:20, 4:20, 5:20), preferably 3:20.
Further, the pharmaceutical composition can prevent or treat liver cancer by causing compensatory up-regulation of SLC7A11 protein expression level.
Furthermore, the pharmaceutical composition can inhibit the activity of System Xc, and lead to compensatory up-regulation of the expression level of SLC7A11 protein so as to prevent or treat liver cancer.
Furthermore, the pharmaceutical composition can increase BECN-SLC 7A11 complex formation, so that the System Xc-activity is inhibited, and the compensatory up-regulation of SLC7A11 protein expression is caused, so that liver cancer can be prevented or treated.
Furthermore, the pharmaceutical composition can activate the AMPK-BECN1 pathway, increase the formation of BECN-SLC 7A11 complex, inhibit the System Xc-activity, and cause compensatory up-regulation of SLC7A11 protein expression level so as to prevent or treat liver cancer.
The present invention provides in a second aspect the use of a pharmaceutical composition as described in the first aspect for the manufacture of a medicament for the treatment of cancer.
Further, the pharmaceutical composition comprises ursolic acid and sulfasalazine.
Further, the mass ratio of ursolic acid to sulfasalazine is 1-5:20 (such as 1:20, 2:20, 3:20, 4:20, 5:20), preferably 3:20.
Further, the cancer is liver cancer.
Furthermore, the medicine can cause compensatory up-regulation of SLC7A11 protein expression quantity to prevent or treat liver cancer.
Furthermore, the medicine can inhibit the activity of System Xc, and lead to compensatory up-regulation of the expression level of SLC7A11 protein so as to prevent or treat liver cancer.
Furthermore, the medicine can increase BECN-SLC 7A11 complex formation, so that the System Xc-activity is inhibited, and the compensatory up-regulation of SLC7A11 protein expression is caused, so that liver cancer can be prevented or treated.
Furthermore, the medicine can activate the AMPK-BECN1 pathway, increase the formation of BECN1-SLC7A11 complex, inhibit the System Xc-activity, and cause compensatory up-regulation of SLC7A11 protein expression level so as to prevent or treat liver cancer.
The invention provides an application of ursolic acid in preparing medicines of the sulfasalazine anti-liver cancer sensitizer in a third aspect, wherein the mass ratio of the ursolic acid to the sulfasalazine is 1-5:20 (such as 1:20, 2:20, 3:20, 4:20 and 5:20), and is preferably 3:20.
Furthermore, the medicine can cause compensatory up-regulation of SLC7A11 protein expression quantity, increase apoptosis of liver cancer cells and prevent or treat liver cancer.
Furthermore, the medicine can inhibit the activity of System Xc, and lead to compensatory up-regulation of the expression level of SLC7A11 protein so as to prevent or treat liver cancer.
Furthermore, the medicine can increase BECN-SLC 7A11 complex formation, so that the System Xc-activity is inhibited, and the compensatory up-regulation of SLC7A11 protein expression is caused, so that liver cancer can be prevented or treated.
Furthermore, the medicine can activate the AMPK-BECN1 pathway, increase the formation of BECN1-SLC7A11 complex, inhibit the System Xc-activity, and cause compensatory up-regulation of SLC7A11 protein expression level so as to prevent or treat liver cancer.
In the invention, structural formulas of ursolic acid and sulfasalazine are respectively shown as formulas (I) and (II):
（Ⅰ）
（Ⅱ）。
Further, the mass concentration of the ursolic acid is 18-22mg/L (such as 18mg/L, 18.5mg/L, 19mg/L, 19.5mg/L, 20mg/L, 20.5mg/L, 21mg/L, 21.5mg/L and 22 mg/L).
Further, the mass concentration of the sulfasalazine is 130-135mg/L (such as 130mg/L, 131mg/L, 132mg/L, 133mg/L, 134mg/L and 135 mg/L).
Further, the mass concentration of the ursolic acid is 20 mg/L.
Further, the mass concentration of the sulfasalazine is 133.33 mg/L.
Further, the daily dosage of the ursolic acid is 30 mg/kg.
Further, the daily dosage of sulfasalazine is 200 mg/kg.
Further, the administration frequency of the ursolic acid is 1 time a day as a unit dose.
Further, the frequency of administration of the sulfasalazine is 1 time per day in unit dose.
Further, the administration sequence of the ursolic acid and the sulfasalazine can be separate administration, simultaneous administration or sequential administration.
Further, the medicament is in the form of injection or oral preparation.
Further, the dosage form of the medicine can be capsules, tablets, granules, powder, pills, dripping pills, sustained and controlled release preparations, oral liquid, mixture, syrup, liquid injection, powder for injection or tablets for injection.
In a fourth aspect, the present invention provides the use of a pharmaceutical composition according to the first aspect for the manufacture of a medicament for the prevention or treatment of liver cancer by causing up-regulation of the SLC7a11 protein.
In a fifth aspect, the present invention provides a use of a pharmaceutical composition according to the first aspect for the preparation of a medicament for preventing or treating liver cancer by inhibiting System Xc-activity.
In a sixth aspect, the present invention provides a use of a pharmaceutical composition according to the first aspect for the preparation of a medicament for increasing BECN-SLC 7a11 complex formation by activating the AMPK-BECN1 pathway, thereby preventing or treating liver cancer.
In the invention, after the ursolic acid and the sulfasalazine are combined, the AMPK-BECN1 pathway is activated, the formation of BECN1-SLC7A11 complex is increased, and the System Xc-activity is inhibited, so that the compensatory up-regulation of the SLC7A11 protein expression is caused.
The invention has the beneficial effects that:
1. The invention discovers that the ursolic acid has the effect of enhancing the iron death of the salazosulfapyridine induced liver cancer cells for the first time, has good synergistic effect on the anti-liver cancer treatment of the salazosulfapyridine, and can increase the iron death of the salazosulfapyridine induced SMMC-7721 human liver cancer cells. The ursolic acid with the concentration of 12 mu M and the sulfasalazine with the concentration of 0.75 mM are added to jointly treat the human liver cancer cell strain SMMC-7721 cells, the cell activity is obviously reduced compared with that of a blank group, and the activity of the human liver cancer cells which are jointly treated by the ursolic acid and the sulfasalazine is only about 40%;
2. The invention discovers that the ursolic acid has good prevention effect on the possible potential hepatotoxic effect of the sulfasalazine for the first time, and the combined treatment of the ursolic acid and the sulfasalazine can obviously reduce the level of glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) from the aspect of serum biochemical indexes, thereby reducing liver injury.
Drawings
FIG. 1 is a graph showing the effect of different treatments on tumor volume change and tumor weight in a model of SMMC-7721 ectopic transplanted mice (wherein, graph A is a schematic diagram of tumor entity state of different agents after treatment of mice, graph B is a graph showing the trend of tumor volume change with time of different agents after treatment of mice, graph C is a graph showing the trend of weight change with time of mice after treatment of mice with different agents, graph D is a graph showing the trend of tumor weight change of different agents after treatment of mice, graph E is the level of alanine aminotransferase ALT in serum of mice after treatment of mice with different agents, and graph F is the level of glutamic oxaloacetic aminotransferase AST in serum of mice after treatment of mice with different agents).
FIG. 2 shows the effect of ursolic acid on iron death of SMMC-7721 human hepatoma cells induced by sulfasalazine (FIG. A shows the levels of activity of different agents on AML12 cells of the normal mouse hepatoma cell line after mouse treatment; FIG. B shows the levels of activity of different agents on AML12 cells of the normal mouse hepatoma cell line after mouse treatment).
FIG. 3 shows molecular mechanisms of ursolic acid for enhancing iron cell death induced by sulfasalazine (wherein, FIG. A shows the uptake capacity of human liver cancer cell line SMMC-7721 cells to cystine after mice are treated by different reagents, FIG. B shows the content of cysteine in human liver cancer cell line SMMC-7721 cells after mice are treated by different reagents, FIG. C shows the Western blotting result of mice treated by different reagents, and FIG. D shows the immunoprecipitation result of mice treated by different reagents).
Detailed Description
In order that the technical content of the present invention may be more clearly understood, the following detailed description of the embodiments is given only for better understanding of the content of the present invention and is not intended to limit the scope of the present invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the reagents, materials, etc. used in the examples described below are commercially available unless otherwise specified.
The term "System xc-" is an antiport protein consisting of the light chain subunit (xCT, SLC7A 11) and the heavy chain subunit (CD 98hc, SLC3A 2), mainly responsible for cellular uptake of cystine in exchange for intracellular glutamate. .
The term "SLC7A11" is the gene name of the System xc-protein, which encodes the xCT subunit of System xc-. SLC7a11 is a transmembrane protein that plays an important role in cystine transport on cell membranes.
The term "BECN-SLC 7A11" refers to the interaction between BECN1 and SLC7A11, BECN1 being a protein involved in the autophagy process, and SLC7A11 being a subunit of the System xc-protein. The formation of BECN-SLC 7A11 complex may result in inhibition of System xc-activity.
The term "AMPK-BECN1" refers to the interaction between AMPK (AMP-ACTIVATED PROTEIN KINASE) and BECN 1. AMPK is a cellular energy metabolism regulating protein kinase, and phosphorylated AMPK can activate BECN A1 to promote BECN-SLC 7a11 formation.
Example 1 Effect of ursolic acid on increasing the anti-liver cancer efficacy of sulfasalazine in mice
The test method comprises the following steps: male BALB/C nude mice (Beijing Vitrehua laboratories Inc.), 24, 5 weeks old, 100 μ LPBS containing 5×10 6 SMMC-7721 cells were mixed with an equal volume of Matrigel, injected subcutaneously on the right side of the mice, and tumor volumes were measured with vernier calipers. When the tumor volume reached 75 mm 3, mice were randomly divided into control, ursolic acid, sulfasalazine and combination. The control group was given the corresponding solvent, the ursolic acid treatment group was given by intraperitoneal injection at a dose of 30 mg/kg per day, the sulfasalazine treatment group was given by intraperitoneal injection at a dose of 200 mg/kg per day, and the combination treatment group was given by intraperitoneal injection of a mixed solution containing the corresponding doses of ursolic acid and sulfasalazine. Mice body weight and tumor volume were measured and recorded every four days. After the experiment was completed, serum was collected, and a part of tumor was stored in 10% formalin, and the remaining liquid nitrogen was flash frozen and stored at-80 ℃.
With the volume and the weight of the tumor as evaluation criteria, it can be seen from fig. 1A that the single action of ursolic acid and sulfasalazine can inhibit the growth of the tumor, but the effect is not obvious, the combined treatment can significantly inhibit the growth of tumor tissues, and as shown in fig. 1B, the inhibition rate of the combined treatment group on the volume of the tumor can reach 55%; compared with the single ursolic acid acting group, the inhibition rate of the combined treatment group on the tumor volume is improved by 38%; compared with the sulfasalazine single acting group, the inhibition rate of the combined treatment group on the tumor volume is improved by 26%; as shown in fig. 1C, during the treatment period, the weights of the mice in different groups are not significantly different and are not significantly reduced, as shown in fig. 1D, the tumor quality can be significantly reduced in the combined treatment group, and the inhibition rate of the tumor quality can reach 55% in the combined treatment group; compared with the single acting group of ursolic acid, the inhibition rate of the combined treatment group on the tumor quality is improved by 35 percent, and compared with the single acting group of sulfasalazine, the inhibition rate of the combined treatment group on the tumor quality is improved by 33 percent; from the aspect of serum biochemical indexes, the combined treatment can also obviously reduce the levels of glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST), and reduce liver injury, as shown in fig. 1E, the ALT level of the combined treatment group is reduced by 60%, the ALT level reduction rate of the combined treatment group is improved by 32% compared with that of an ursolic acid single acting group, and the ALT level reduction rate of the combined treatment group is improved by 26% compared with that of an sulfasalazine single acting group. As shown in fig. 1F, the AST level of the combined treatment group was reduced by 40%, the AST level of the combined treatment group was increased by 30% as compared with the ursolic acid alone, and the AST level of the combined treatment group was increased by 23% as compared with the sulfasalazine alone. .
In conclusion, in the subcutaneous tumor model of the SMMC-7721 liver cancer of the nude mice, the combined treatment of the ursolic acid and the sulfasalazine has good treatment effect on the liver cancer.
Example 2 Ursolic acid increased iron death of Sulfosamine-induced SMMC-7721 human liver cancer cells
After the human liver cancer cell line SMMC-7721 cells 36 h are treated by the ursolic acid with the low dosage concentration of 12 mu M and the sulfasalazine with the concentration of 0.75 mM singly or in combination, the cell activity is detected by a crystal violet staining method. The results shown in fig. 2 demonstrate that the cellular activities of ursolic acid alone and salazosulfapyridine alone were not significantly different from the control group, whereas the combined treatment group significantly reduced the cellular activities. After 3 h of pretreatment with iron death inhibitor Fer 1 or Lip 1, the combined treatment with ursolic acid and sulfasalazine increased the cell activity significantly compared to the combined treatment without pretreatment with iron death inhibitor. The result shows that the ursolic acid can increase the iron death of SMMC-7721 human liver cancer cells induced by the salazosulfapyridine. The ursolic acid also has the same sensitization effect on the iron death inducer erastin which has the same action target point as the sulfasalazine. As shown in FIGS. 2A and 2B, the combined treatment further enhanced the decrease in cell activity caused by the erastin, and this cell activity inhibition was rescued by the iron death inhibitor, and as shown in FIG. 2A, the inhibition of cell activity was about 9% in the ursolic acid alone group, about 10% in the sulfasalazine alone group, and about 57% in the combined treatment group, and about 40% in the combined treatment after pretreatment with the iron death inhibitor Fer 1 or Lip 1. Similarly, the inhibition rate of the single treatment of the erastin on the cell activity is about 27%, the inhibition rate of the single treatment of the erastin on the cell activity is increased to 69% after the combined treatment of the erastin and the ursolic acid, and the inhibition rate of the single treatment of the erastin on the cell activity is respectively reduced to 30% and 34% after the rescue of the Fer 1 or the Lip 1. In contrast, for the normal hepatocyte line AML12 cells of mice, the same concentrations of ursolic acid and sulfasalazine treated alone or in combination did not cause significant decrease in cell activity, and as shown in FIG. 2B, the sulfasalazine treated alone or in combination with ursolic acid did not show significant difference in cell activity from the control group. This suggests that liver cancer cells are more sensitive to the effects of ursolic acid sensitization by sulfasalazine on induction of cellular iron death than normal liver cells.
After the human liver cancer cell line SMMC-7721 cells 3 h are treated by the ursolic acid with the low dosage concentration of 12 mu M and the sulfasalazine with the concentration of 0.75 mM singly or in combination, the cells are collected, and the cystine uptake capacity and the intracellular cysteine content are measured. As shown in fig. 3A, 3B, 3C and 3D, the combined treatment resulted in a further decrease in the cystine uptake capacity of the cells and the intracellular cysteine content, indicating that the System Xc-activity was significantly inhibited, as shown in fig. 3A, the cystine uptake capacity of the cells in the sulfasalazine alone group was decreased to about 70%, the cystine uptake capacity of the cells in the ursolic acid alone group was decreased to about 90%, and the cystine uptake capacity of the cells in the combined treatment was decreased to about 40%, demonstrating that the combined treatment resulted in a significant decrease in the cystine uptake capacity of the cells; as shown in FIG. 3B, the intracellular cysteine content in the sulfasalazine alone group was reduced to about 50%, the intracellular cysteine content in the ursolic acid alone group was reduced to about 70%, and the intracellular cysteine content in the combination treatment was reduced to about 30%, demonstrating that the combination treatment resulted in a substantial reduction in intracellular cysteine content. The results of western blotting and immunoprecipitation show that when ursolic acid and sulfasalazine are combined, the AMPK-BECN1 pathway is activated, the formation of BECN1-SLC7A11 complex is increased, the System Xc-activity is inhibited, the compensatory up-regulation of SLC7A11 protein expression is caused, and as shown in figure 3C, the protein expression levels of SLC7A11, P-AMPK alpha and P-BECN1 in the combined treatment group are obviously increased compared with that of the sulfasalazine alone treatment group. As shown in FIG. 3D, the level of BECN-SLC 7A11 complex in the combination treatment group was further increased compared to that of the sulfasalazine alone and the ursolic acid treatment group.
Claims (4)
1. The pharmaceutical composition consists of ursolic acid and sulfasalazine, and the structural formulas of the ursolic acid and the sulfasalazine are shown as the formulas (I) and (II) respectively:
（Ⅰ）
（Ⅱ）；
The pharmaceutical composition can prevent or treat liver cancer by causing compensatory up-regulation of SLC7A11 protein expression level; in the pharmaceutical composition, the mass ratio of the ursolic acid to the sulfasalazine is 3:20.
2. Use of the pharmaceutical composition according to claim 1 for the preparation of a medicament for preventing or treating liver cancer.
3. The use of claim 2, wherein the agent is capable of activating the AMPK-BECN1 pathway, increasing formation of the BECN-SLC 7a11 complex, resulting in inhibition of System Xc-activity, resulting in compensatory up-regulation of SLC7a11 protein expression.
4. The use according to claim 2 or 3, wherein the medicament is in the form of an injection or an oral preparation.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103142620A (en) * | 2013-03-18 | 2013-06-12 | 中国科学院新疆理化技术研究所 | Application of ursolic acid to antitumor immunity |
| CN109674789A (en) * | 2019-01-23 | 2019-04-26 | 中国医学科学院基础医学研究所 | The purposes of carboxyltriazole and glutamate uptake and metabolic poison in antitumor |
| CN109908173A (en) * | 2019-03-25 | 2019-06-21 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A kind of sulfasalazine iron complex application in preparation of anti-tumor drugs |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103142620A (en) * | 2013-03-18 | 2013-06-12 | 中国科学院新疆理化技术研究所 | Application of ursolic acid to antitumor immunity |
| CN109674789A (en) * | 2019-01-23 | 2019-04-26 | 中国医学科学院基础医学研究所 | The purposes of carboxyltriazole and glutamate uptake and metabolic poison in antitumor |
| CN109908173A (en) * | 2019-03-25 | 2019-06-21 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A kind of sulfasalazine iron complex application in preparation of anti-tumor drugs |
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