WO2017011973A1 - Use of statin compound for treating stomach cancers - Google Patents

Abstract

Provided in the present invention is a use of a statin compound for preparing pharmaceutical compositions for treating stomach cancers. The compound can induce the apoptosis of stomach cancer cells, inhibit the growth and metastasis of stomach cancer cells, and negatively regulate transcription factors related to the development of stomach cancer.

Description

Use of statins for the treatment of gastric cancer Technical field

The invention relates to a second use of a compound, in particular to the use of a statin for the treatment of gastric cancer.

Background technique

According to the statistics of the cause of death in 2013 by the Ministry of Health and Welfare, cancer is the top ten cause of death, and gastric cancer is the fifth leading cause of cancer death. Gastric cancer is characterized by mucosal lesions in the stomach, which produce ulcers, which in turn cause malignant tumor cells to form in the stomach. The cells in the stomach will not naturally undergo apoptosis, and will continue to undergo cell division and proliferation, and the gastric cancer cells will simultaneously invade the growth range of normal cells during the process of proliferation, and then gradually invade the stomach and other organs, causing the cancer cells to migrate. Since the majority of gastric cancer patients have a distal part of the stomach, and gastric cancer patients have no specific clinical symptoms, most patients with gastric cancer are diagnosed with advanced gastric cancer. Therefore, the mortality rate of gastric cancer has not been reduced. .

At present, the clinical treatment of gastric cancer includes surgical resection, chemotherapy, radiation therapy, and targeted drugs. Among them, the most important treatment is surgery, even for patients who cannot be cured by surgery, they will still be palliative. Excision to slow tumor growth and prevent tumor metastasis. Chemotherapy achieves the goal of killing or controlling cancer cells by administering appropriate anticancer agents to patients. However, current research indicates that chemotherapy is not effective for gastric cancer, and the efficacy of treatment with a single drug is only 20 %, even with multiple drugs, the expected therapeutic effect is not achieved. Radiation therapy causes X-rays or particles to kill cancer cells. In general, radiation therapy is an adjuvant therapy, which is used to remove cancer cells that cannot be removed, residual or invisible to the naked eye, and can only achieve local control of cancer cells. Targeted drugs are the focus of current treatments for gastric cancer. The current study found that for patients with gastric cancer specimens with HER-2 positive staining, the efficacy of HER-2 target drugs is better than traditional chemotherapy, but the target drugs cannot. Adapting to all patients with gastric cancer, and the high cost of medical care, many cancer patients can not afford the drug and miss treatment.

It can be seen from the above that there is still a lack of an effective and economical treatment for gastric cancer. It takes a lot of money and time to develop based on new drugs, and the development success rate is low, so that new drug development is not easy, and even if a new drug is successfully developed, patients need to pay a higher treatment fee to use the new drug. Therefore, in order to reduce the cost of drug development and improve the development efficiency, it is an important subject of current researchers to screen out anti-gastric cancer pharmaceutical compositions with high safety and good efficacy from existing drugs.

Summary of the invention

The main object of the present invention is to provide a use of a statin compound for the manufacture of a pharmaceutical composition for treating gastric cancer, which can be used clinically effectively and safely.

Another object of the present invention is to provide a use of a statin compound for reducing the development of a drug. Ben, and save time in drug development.

In order to achieve the above object, the present invention selects a statin having an activity for treating gastric cancer from an existing drug, and can effectively improve or treat gastric cancer by administering an effective amount of a statin compound to an individual suffering from gastric cancer.

In the embodiments of the present invention, a use of a statin for the manufacture of a pharmaceutical composition for treating gastric cancer is disclosed, wherein the statin compound can effectively inhibit gastric cancer cell growth, migration and invasion, and induce gastric cancer cells. Towards apoptosis, negative regulation of transcription factors associated with the development of gastric cancer. Therefore, the pharmaceutical composition containing the statin compound disclosed in the present invention does have an effect of treating gastric cancer.

Preferably, the statin is lovastatin, simvastatin, fluvastatin, atorvastatin, pravastatin, rosuvastatin Rosuvastatin or pitavastatin.

DRAWINGS

Figure 1 shows the cell viability of AGS, MKN45 and TSGH9201 gastric cancer cell lines treated with different concentrations of statins.

Figure 2A shows the cellular activity of AGS gastric cancer cell lines treated with statins at different times.

Figure 2B shows the cell viability of MKN45 gastric cancer cell lines treated with statins at different times.

Figure 2C shows the cellular activity of the TSGH9201 gastric cancer cell line treated with statins at different times.

Figure 3A shows the results of flow cytometry analysis of AGS gastric cancer cell lines after treatment with different concentrations of statins.

Figure 3B shows the results of measuring the number of cells in a MKN45 gastric cancer cell line treated with different concentrations of statins by flow cytometry.

Figure 3C shows the results of measuring the number of cells in a TSGH9201 gastric cancer cell line treated with different concentrations of statins by flow cytometry.

Figure 4A shows the results of TUNEL detection of apoptosis in AGS gastric cancer cell lines treated with different concentrations of statins.

Figure 4B shows the results of TUNEL detection of apoptosis in MKN45 gastric cancer cell lines treated with different concentrations of statins.

Figure 4C shows the results of TUNEL detection of apoptosis in TSGH9201 gastric cancer cell lines treated with different concentrations of statins.

Fig. 5 shows the expression of AGS, MKN45 and TSGH9201 gastric cancer cells treated with statins at different times by Western blotting, and the expression of proteins regulating gastric cancer cell apoptosis in each gastric cancer cell was detected.

Figure 6A shows the different treatments of each group of AGS gastric cancer cells cultured in transwell medium for 12 hours. The results of cell transfer of AGS gastric cancer cells in each group were observed and analyzed.

Fig. 6B shows the results of cell migration of each group of MKN45 gastric cancer cells after different treatments of each group of MKN45 gastric cancer cells cultured in transwell medium for 12 hours.

Fig. 6C shows the results of cell transfer of each group of TSGH9201 gastric cancer cells after differently treated groups of TSGH9201 gastric cancer cells cultured in transwell medium for 12 hours.

Fig. 7A shows the results of cell transfer of each group of AGS gastric cancer cells after 16 hours of culture in a colloidal medium by different treatments of each group of AGS gastric cancer cells.

Fig. 7B is a result of analyzing the cell transfer of each group of MKN45 gastric cancer cells after being cultured for 16 hours in colloidal medium by differently treated groups of MKN45 gastric cancer cells.

Fig. 7C is a result of analyzing the cell transfer of each group of TSGH9201 gastric cancer cells after being cultured for 12 hours in colloidal medium by differently treated groups of TSGH9201 gastric cancer cells.

Fig. 8A shows the results of fluorescent staining of gastric cancer cells of different groups of AGS treated with different treatments, and the results of fluorescence staining of gastric cancer cells of each group were observed by confocal microscopy.

FIG. 8B shows the results of fluorescent staining of each group of MKN45 gastric cancer cells treated with different treatments, and the results of fluorescence staining of each group of MKN45 gastric cancer cells were observed by confocal microscopy.

8C shows the results of fluorescent staining of gastric cancer cells of different groups of TSGH9201 treated with different treatment, and the results of fluorescence staining of gastric cancer cells of each group were observed by confocal microscopy.

Fig. 9 shows the results of detecting the gastric cancer cells of each group treated with different conditions for 12 hours by Rho protein activity assay.

Figure 10 is a graph showing the results of detecting the activity of matrix metalloproteinase-2 in each group of gastric cancer cells by gelatin matrix metalloproteinase activity assay.

Figure 11 shows the expression of phosphorylated SATA3, vimentin, transcription factor Snail and transcription factor Twist in each group of gastric cancer cells treated with statins at different times by Western blotting.

Figure 12 is a graph showing the expression of phosphorylated SATA3, vimentin, transcription factor Snail and transcription factor Twist in each of the gastric cancer cells treated with different conditions by Western blotting.

Figure 13 shows the statistical results of total mortality of gastric cancer between regular users and non-users of statins.

detailed description

The invention discloses the use of a statin, in particular, because the statin has the ability to inhibit the growth, metastasis and invasion of gastric cancer cells, and can cause gastric cancer cells to apoptosis, therefore, by administering A pharmaceutical composition containing an effective amount of a statin to a gastric cancer patient can achieve the efficacy of manufacturing a gastric cancer.

The meaning of the technical and scientific terms used in the description and claims of the present invention are the same as those of ordinary skill in the art. If there is contradiction In case of matter, the content of the present invention shall prevail.

The so-called "statin compound" is a drug currently used clinically to treat hyperlipidemia, mainly by inhibiting the activity of key enzymes in cholesterol synthesis in hepatocytes, reducing cholesterol production, and reducing the incidence of coronary heart disease. And mortality. Compared with other hypolipidemic drugs, statins have fewer side effects. According to the statistics of existing studies, only 5% of patients have obvious side effects. Therefore, statins are considered to be highly safe. Pharmaceutical composition. There are many types of statins currently in clinical use, including lovastatin, simvastatin, fluvastatin, atorvastatin, and pravastatin (pravastatin). ), rosuvastatin, pitavastatin, and the like. The mechanism of action based on statins is similar, so that simvastatin is exemplified in the following examples, but this is not intended to limit the scope of the claims.

Furthermore, according to the current research data, the frequency of clinical use of statins is initially used once a day, the dose is 2 ~ 80 mg, and the frequency of subsequent continuous use is once a day, the dose is 1 ~ 80 mg. For example, the dosage of different statins will vary, as shown in Table 1 below.

Table 1: Dosage of statins

By "pharmaceutical composition" is meant an effective amount of the desired compound or active ingredient to produce a particular effect, and at least one pharmaceutically acceptable carrier. As is known to those of ordinary skill in the art to which the present invention pertains, the dosage form of the pharmaceutical composition varies depending on the manner in which the particular effect is to be caused, such as a lozenge, a powder, an injection, etc., and the carrier also follows the medicine. The dosage form of the composition is obtained as a solid, semi-solid or liquid. For example, carriers include, but are not limited to, gelatin, emulsifiers, hydrocarbon mixtures, water, glycerin, physiological saline, buffered saline, lanolin, paraffin, beeswax, dimethicone, ethanol.

By "effective amount" is meant an amount of the active ingredient or compound to be produced in a pharmaceutical composition to produce the desired effect, usually expressed as a percentage by weight of the active ingredient or compound in the pharmaceutical composition. As is known to those of ordinary skill in the art to which the present invention pertains, the effective amount will be due to the administration of a particular effect. The way it is different. Generally, the active ingredient or compound will be present in the compositions in an amount of from about 1% to about 100%, preferably from about 30% to about 100% by weight of the composition.

In the following, in order to explain the advantages of the present invention, the embodiments of the present invention are described in detail. Scope and meaning.

Example 1: Effect of statins on cancer cell activity

Three gastric cancer cell lines: AGS, MKN45 and TSGH9201 were taken and cultured separately, and each of the gastric cancer cell lines was treated with simvastatin at concentrations of 0, 1.25, 2.5, 5, 10 and 20 μg/ml for 72 hours. . The activity of each of the gastric cancer cell lines treated with different concentrations of simvastatin was analyzed by a cell activity staining assay (MTS assay), and the results are shown in Fig. 1. Further, the three gastric cancer cell lines were respectively divided into two groups, wherein the first group did not treat simvastatin; the second group was treated with simvastatin at a concentration of 20 μg/ml for 72 hours. The cell viability of the first and second groups of each of the gastric cancer cell lines was analyzed by cell activity staining at 0, 24, 48 and 72 hours after simvastatin treatment, respectively, and the results are shown in Fig. 2.

Furthermore, each of the gastric cancer cell lines treated with different concentrations (0, 1.25, 2.5, 5, 10, and 20 μg/ml) of simvastatin was collected by flow cytometry (FCM) and TUNEL detection, respectively. The method (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analyzed the DNA content and apoptosis of each of the gastric cancer cell lines treated with different concentrations of simvastatin, and the results are shown in Fig. 3 and Fig. 4, respectively.

The results of Fig. 1 and Fig. 2 show that statins such as simvastatin can actually reduce the activity of gastric cancer cells, and when the concentration of statin treatment is increased or the treatment time is increased, the activity of gastric cancer cells will also increase. low. As can be seen from the results of FIG. 3 and FIG. 4, statin compounds such as simvastatin can induce apoptosis of gastric cancer cells, thereby reducing the activity of gastric cancer cells and inhibiting the growth of gastric cancer cells.

The results of FIGS. 1 to 4 show that the statin compound of the present invention has an ability to inhibit the growth of gastric cancer cells and induce apoptosis of gastric cancer cells, and can be used as an active ingredient in a pharmaceutical compound for treating or slowing down gastric cancer and its symptoms.

Example 2: Effect of statins on apoptosis of cancer cells

The three gastric cancer cell lines: AGS, MKN45 and TSGH9201 were treated with simvastatin at a concentration of 20 μg/ml, respectively, and cultured at 0, 2, 6, 12 and 24 hours, respectively. Protein-specific analysis was performed by Western blotting to observe proteins regulating gastric cancer cell apoptosis in each of the gastric cancer cell lines: MCL-1, BAX, Bcl-2, Bcl-xL, NOXA, PUMA, lytic protease -3 (cleaved caspase-3) and the performance of cleavage PARP (cleaved PARP), The results are shown in Fig. 5, in which β actin was a control group.

From the results in Figure 5, simvastatin can inhibit the expression of anti-apoptotic proteins: Bcl-2, Bcl-xL and Mcl-1L, and at the same time promote the promotion of apoptotic proteins: BAX, NOXA, PUMA and Mcl-1S. Markers that express and increase apoptosis: Apoptosis proteinase-3 and PARP.

From the above results, the statin of the present invention can regulate the apoptosis of gastric cancer cells through the mitochondrial pathway in gastric cancer cells, and can make the gastric cancer cells go to apoptosis and achieve the effect of treating gastric cancer.

Example 3: Effects of statins on cancer cell metastasis and erosion

The three gastric cancer cell lines: AGS, MKN45 and TSGH9201 were divided into four groups, of which the first group was a blank group; the second group was treated with simvastatin at a concentration of 5 μg/ml; the third group was treated with 3 mM. Cells were treated with mevalonate; the fourth group was simultaneously treated with 5 μg/ml of simvastatin and 3 mM mevalonate. Each of the gastric cancer cell lines treated under different conditions was cultured in a cell migration transwell medium for 12 hours to observe the metastasis of gastric cancer cells, and the results are shown in Fig. 6. In addition, each of the gastric cancer cell lines treated under different conditions was cultured in a colloidal medium for 16 hours to observe gastric cancer cell erosion, and the results are shown in FIG.

It is known from the prior art that fibroactin (F-actin) is one of the cytoskeletal components and an important physiological component for the transfer of cancer cells. Therefore, it is necessary to analyze the migration of cancer cells by observing fibronectin in the cells. . Therefore, after treating the first to fourth groups of each of the gastric cancer cell lines for 12 hours under the treatment conditions thereof, the cytoskeleton and the DAPI dye were labeled with rhodamine-labeled phalloidin. The nuclei were stained, fluorescent staining was performed, and the results of fluorescence staining were observed by confocal microscopy, as shown in Fig. 8. Among them, blue was the result of nuclear staining, and red was the result of fibronectin (F-actin) staining. .

It is disclosed in the prior art that the Rho protein belongs to a small G protein family, has an activated state bound to GDP and an inactive state bound to GTP, and the excessive expression of Rho protein is closely related to tumor development and degree of malignancy, and thus By observing the expression of Rho protein in cells, it can be used to analyze the degree of development of tumor cells. Therefore, each group of gastric cancer cells treated under different conditions for 12 hours was examined by Rho protein activity assay, and the results are shown in FIG.

Furthermore, each of the gastric cancer cell lines of the first to fourth groups was treated under different conditions, cultured in a serum-free medium for 24 hours, and then detected by a gelatin matrix metalloproteinase assay (Gelatin zymogram assay). The activities of matrix metalloproteinase-9 and matrix metalloproteinase-2 in gastric cancer cells, the activity of matrix metalloproteinase 2 in each group of gastric cancer cells is shown in Fig. 10. Since matrix metalloproteinase-9 and matrix metalloproteinase 2 are considered to be associated with tumor metastasis, it is possible to detect the turnover of simvastatin on tumor cells by observing the activity of matrix metalloproteinase-9 and matrix metalloproteinase-2 in cells. influences.

From the results of Fig. 6 and Fig. 7, it can be seen that the phenomenon of gastric cancer cell migration and invasion actually decreased in the second group treated with simvastatin compared with the other three groups. From the results of Fig. 8, it was found that in the first group, the third group and the fourth group, the cytoskeleton of the gastric cancer cells was filiformly staggered, and the appearance of the gastric cancer cells of the second group was significantly changed compared with the other groups. The cytoskeletal structure is looser and the staining is lighter, and it is obvious that the cytoskeletal content is less.

Further, from the results of Fig. 9, it was revealed that the activation of RhoA-GTP was inhibited by the second group treated with simvastatin, and the inactivation of RhoA-GDP was further enhanced. As can be seen from the results of FIG. 10, in the second group treated with simvastatin, matrix metalloproteinase 2 in gastric cancer cells was significantly inhibited, and the inhibitory effect of simvastatin-treated AGS gastric cancer cells was the most remarkable.

From the results of FIGS. 6 to 10, it can be seen that the statin of the present invention can inhibit the growth of gastric cancer cells and inhibit the invasion of other cells by inhibiting the growth of gastric cancer cells. Accordingly, the statins of the present invention have the efficacy of treating or ameliorating gastric cancer and related symptoms.

Example 4: statins can negatively regulate the epithelial transition marker (EMT marker)

Since the transcription factors SATA-3 and Twist are key regulators of gastric cancer cell development, the three gastric cancer cell lines: AGS, MKN45 and TSGH9201 were treated with simvastatin at a concentration of 5 μg/ml, respectively, 0, 6, and 12. After 24 hours, the expression of phosphorylated SATA3, vimentin, transcription factor Snail, and transcription factor Twist in each of the cells treated with simvastatin for different times was measured by Western blotting, and the results are shown in FIG.

Further, the three gastric cancer cell lines: AGS, MKN45, and TSGH9201 were respectively divided into four groups, and the treatment conditions of each of the groups were as described in Example 3, and the gastric cancer cells were treated according to the treatment conditions of each of the groups. After an hour, the expression of phosphorylated SATA3, vimentin, transcription factor Snail, and transcription factor Twist in each group of gastric cancer cells was determined by Western blotting, and the results are shown in FIG.

As can be seen from Fig. 11, simvastatin can reduce the expression of transcription factors regulating the development of gastric cancer cells, and as the treatment time increases, the expression of transcription factors also decreases. From the results of FIG. 12, it was found that the expression factors of the gastric cancer cells related to the development of gastric cancer in the first group, the third group, and the fourth group did not decrease. In contrast, the transcription factors associated with the development of gastric cancer in the second group of gastric cancer cells were significantly decreased.

From the above results, it is revealed that the statin compound disclosed in the present invention can inhibit the continued development of gastric cancer cells, and can achieve the efficacy of treating or improving gastric cancer and related diseases.

Example 5: Statistical analysis

Taking gastric cancer from the admission diagnosis data of the National Health Insurance Research Database (NHIRD) and the registered database of major injured patients The patient's data was analyzed according to the following procedures: First, the patients who were newly diagnosed with gastric cancer and registered in the database of major patients with injuries were selected from January 1, 1997 to December 31, 2011. There are 44,259 people in total. A total of 44,259 patients who met the above qualifications were identified, and a total of 15,582 patients who had not undergone gastric surgery and 166 patients who had undergone non-gastric cancer surgery were selected. Therefore, patients who had undergone gastric cancer treatment and had undergone gastric cancer surgery were screened. A total of 28,511. From the 28,511 patients excluded from the gastric cancer surgery, patients diagnosed with other tumors, leaving 26,806 patients, and then excluded patients with tracking time less than 3 months, and finally left 25010 patients. The 25010 patients were divided into three groups according to the frequency of their administration of statins. Among them, there were 22867 patients in the non-use group; 1176 patients in the regular use group; and 967 patients in the use group. After using the statin tendency and continuing to administer the drug for 3 months, the regular use group and the non-frequent use group were distributed in a ratio of 1:4, and the non-use group was divided into 4484 patients, and the frequently used group had 1121 patients, among which, Patient data are shown in Table 2 below. The results of the ongoing statistical analysis of the above patients are shown in Figure 13 and Tables 3 and 4 below, showing that the overall survival rate of patients who frequently used statins after 8 years was 61.08%, rather than patients who used statins. The overall survival rate after eight years is 47.07%. Among them, the dosage of statins is shown in Table 1 above.

Table 2: Statistical analysis of patient data

Table 3: Results of multivariate analysis

	Risk ratio	95% confidence interval	P value
				Frequently used group	0.45	0.39-0.51	0.00
male	0.99	0.90-1.09	0.86
				Surgical age	1.03	1.02-1.03	0.00
Acute coronary syndrome	1.34	1.19-1.50	0.00
				Brain stroke	1.25	1.08-1.44	0.00
Chronic lung obstruction	1.00	0.85-1.18	0.98
				diabetes	1.89	1.72-2.09	0.00
Renal failure	1.69	1.34-2.13	0.00
				hypertension	1.32	1.18-1.46	0.00
High cholesterol	1.10	0.86-1.39	0.45
				Peptic ulcer	1.03	0.93-1.15	0.55

Table 4: Statistical analysis results

Number of years of survival	0	2	4	6	8
						Non-use group (person)	4484	2225	1077	490	154
Frequent use group (person)	1121	712	377	190	56

The results of the above various examples show that the pharmaceutical composition containing the statin compound of the present invention can indeed treat or improve gastric cancer and/or its related symptoms, and the present invention is compared with the prior art. The disclosed pharmaceutical composition can not only reduce the production cost, but also provide better prognosis survival rate and survival years for gastric cancer patients.

The present invention has been described in detail by the embodiments of the present invention. It should be understood that the appended claims Han photo.

Claims (7)

1. A use of a statin for the manufacture of a pharmaceutical composition for the treatment of gastric cancer and/or a disorder thereof.
2. The use according to claim 1, wherein the statin compound is selected from the group consisting of lovastatin, simvastatin, fluvastatin, atorvastatin, and paclitaxel. A group consisting of pravastatin, rosuvastatin, and pitavastatin.
3. The use according to claim 1, wherein the statin compound has an initial dose of from 2 to 80 mg/day and a maintenance dose of from 1 to 80 mg/day.
4. The use according to claim 1, wherein the statin compound inhibits the growth of gastric cancer cells.
5. The use according to claim 1, wherein the statin compound induces apoptosis of gastric cancer cells.
6. The use according to claim 1, wherein the statin compound negatively regulates a transcription factor associated with the development of gastric cancer.
7. The use according to claim 1, wherein the statin compound inhibits gastric cancer cell metastasis.

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