Application of Statine in preparation of anti-colorectal tumor drugs

Abstract

The invention discloses application of Statine in preparation of anti-colorectal tumor drugs, and belongs to the technical field of pharmacy. The Statine can activate caspase-3 to induce apoptosis by up-regulating expression of tumor cells CCL5, so that colorectal cancer cells such as HT-29, Caco-2, MC38 and the like are remarkably inhibited, and the activity of normal colonic epithelial cells NCM460 is not influenced. Animal experiments show that Statine can effectively inhibit growth of colorectal tumors of MC38 tumor-bearing mice and mice induced by AOM / DSS, and has no obvious toxic or side effect. The invention provides a novel candidate medicine with high efficiency and low toxicity, which is suitable for treating the colorectal cancer, the rectal cancer, the colorectal adenocarcinoma and the colon cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmacy, and particularly relates to application of Statine in preparation of an anti-colorectal tumor drug. BACKGROUND

[0002] In recent years, the incidence and mortality of malignant tumors (cancers) continue to rise, and have become one of the major diseases threatening human health worldwide. According to statistics of the World Health Organization (WHO), cancer is the second leading cause of death worldwide, causing millions of deaths each year. Despite the continuous progress of medical technology, the treatment of cancer still faces great challenges, especially the heterogeneity, metastasis and drug resistance of tumors, which makes the effect of many patients limited in the treatment process.

[0003] At present, the treatment methods of cancer mainly include surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy, etc. Among them, chemotherapy as a traditional treatment method, although it can inhibit tumor growth to a certain extent, has poor selectivity, large toxic and side effects, and easily leads to drug resistance of tumor cells. Targeted therapy and immunotherapy have achieved remarkable results in some types of cancer, but their application range is limited, and some patients still have ineffective treatment or recurrence.

[0004] Therefore, there is still a need in the art to develop more new anti-tumor drugs, especially drugs with high efficiency, low toxicity and broad-spectrum anti-tumor activity. SUMMARY

[0005] In order to overcome the deficiencies of the prior art, the application provides application of (3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid (Statine) in preparation of an anti-colorectal tumor drug.

[0006] To achieve the above-mentioned purpose, the application provides the following technical solutions:

[0007] Application of Statine in preparation of an anti-colorectal tumor drug. The structural formula of the Statine is as follows:

[0008] .

[0009] The application up-regulates the expression of tumor necrosis factor (TNF) signaling pathway, especially CC class chemokine ligand 5 (CCL5) in tumor cells, thereby promoting apoptosis of tumor cells, and has no obvious toxic and side effects, and can be used for preparation of a drug for preventing and / or treating tumors.

[0010] Further, it also includes application of a pharmaceutically acceptable salt, stereoisomer, solvate, crystal form, isotopically labeled or prodrug of the Statine in preparation of an anti-colorectal tumor drug.

[0011] Further, the colorectal tumor includes cecum cancer, rectal cancer, colorectal adenocarcinoma and colon cancer.

[0012] Further, the anti-colorectal tumor drug further includes an excipient.

[0013] Further, the Statine can prevent and / or treat colorectal tumor by up-regulating the expression of chemokine CCL5 in colorectal tumor cells, activating cleaved caspase-3 to promote colorectal tumor cell apoptosis.

[0014] An anti-colorectal tumor drug, wherein the Statine is used as an effective ingredient to form a drug composition with a pharmaceutically acceptable carrier or excipient.

[0015] The combination of Statine and an anti-tumor agent for use in the preparation of an anti-colorectal tumor drug.

[0016] The Statine for use in the preparation of a drug for up-regulating the expression level of chemokine CCL5.

[0017] Compared with the prior art, the present application has the following advantages and technical effects:

[0018] The present application is verified by a variety of cell model tests, tumor-bearing mice and azoxymethane / dextran sulfate sodium (AOM / DSS) induced tumor model tests in mice. At the cellular level, Statine can effectively promote the apoptosis of a variety of colorectal tumor cells (HT-29, Caco-2 and MC38) without affecting the activity of normal colon epithelial cells NCM460 cells. At the individual level, Statine can effectively slow down the tumor growth in tumor-bearing mice and AOM / DSS induced colorectal model mice. At the molecular level, Statine can up-regulate the expression of chemokine CCL5 in tumor cells and further promote the activation of caspase-3 protein to activate apoptosis, proving that Statine can prevent / treat tumors by activating tumor cell apoptosis. The present application verifies that Statine can be applied to the preparation of drugs for preventing and / or treating tumors, thereby providing a feasible solution for preventing / treating tumor diseases in clinical medicine. BRIEF DESCRIPTION OF DRAWINGS

[0019] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and the illustrative embodiments thereof and their descriptions serve to explain the present application, and do not constitute improper limitations on the present application. In the drawings:

[0020] Figure 1Statine inhibits the growth of tumor cells and does not affect the activity of normal colon epithelial cells NCM460; wherein a is the cell viability of HT-29, NCM460, MC38 and Caco-2 cells treated with different concentrations of Statine and Mevastatin for 24 h; b is the colony formation ability of HT-29 and NCM460 cells treated with 320 μM of Statine for 24 h; c is the Western blot analysis of protein expression of HT-29 cells treated with 320 μM of Statine for 24 h; d is the immunofluorescence staining result of cleaved caspase-3 of HT-29 cells treated with 320 μM of Statine for 24 h; e is the survival rate of HT-29 cells transfected with siR-NC or siRNA-CCL5 and then treated with 320 μM of Statine for 24 h;

[0021] Figure 2 Statine inhibits tumor growth in tumor-bearing mice, wherein a is the modeling of tumor-bearing mice and the schematic diagram of Statine administration; b is the image of MC38 tumor after treatment of STA group and PBS group; c is the quantitative analysis and counting of MC38 tumor volume after treatment of STA group and PBS group; d is the HE staining result of MC38 tumor after treatment of STA group and PBS group; e is the immunohistochemical analysis of Ki67 in MC38 tumor after treatment of STA group and PBS group; f is the immunohistochemical analysis of cleaved caspase-3 in MC38 tumor tissue after treatment of STA group and PBS group;

[0022] Figure 3 Statine inhibits tumor growth in AOM / DSS-induced mouse colorectal tumor model, wherein a is the mouse colorectal tumor model established by AOM / DSS and STA treatment; b is the image of mouse colorectum dissection; c is the quantitative analysis of mouse tumor load; d is the statistical data of mouse organ index. DETAILED DESCRIPTION

[0023] The detailed description set forth below will present various example embodiments of the present application, and should not be considered as limiting the scope of the application, but merely as presenting certain aspects, features and embodiments of the application.

[0024] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values, for example, one having ordinary skill in the art with the benefit of this disclosure would understand that it is contemplated that each and every value within the range is to be specifically identified as an independent alternative.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0026] Many modifications and variations of this application of the present application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0027] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0028] The application provides a use of Statine in the preparation of an anti-colorectal tumor drug, and also includes a use of a pharmaceutically acceptable salt, stereoisomer, solvate, crystal form, isotopically labeled or prodrug of the Statine in the preparation of an anti-colorectal tumor drug.

[0029] The colorectal tumor includes cecal cancer, rectal cancer, colorectal adenocarcinoma and colon cancer.

[0030] The anti-colorectal tumor drug prepared by using Statine, wherein the Statine is used as an effective component, and a pharmaceutically acceptable carrier or excipient is used to form a pharmaceutical composition.

[0031] The Statine can be used in combination with an anti-tumor agent to prepare an anti-colorectal tumor drug.

[0032] The Statine can also be used to prepare a drug for up-regulating the expression level of a chemokine CCL5.

[0033] The raw materials used in the application are all purchased from the market.

[0034] The technical solutions of the present application are further illustrated by the following examples.

[0035] Example 1

[0036] Statine (STA) is a product known in the prior art, obtained by direct chemical synthesis. The Statine used in this example was purchased from Glpbio, USA, product code GC32417.

[0037] Performance test:

[0038] 1. Determination of the growth inhibition effect of Statine on tumor cells and normal colon epithelial cells (NCM460)

[0039] Test method:

[0040] Cytotoxicity test:

[0041] Experimental group: HT-29 (human colon cancer cells), NCM460, MC38 (mouse colon cancer cell line), and Caco-2 (human colorectal adenocarcinoma cells) cells were inoculated into 96-well plates, respectively. After the cells were normally attached and grew to 80%, 10 μL of different concentrations of Statine (final concentrations in the wells were 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM, respectively) and 10 μL of different concentrations of Mevastatin (final concentrations in the wells were 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM, respectively) were added for 24 h. The control group (CON) was treated with 10 μL of PBS for 24 h. After 24 h, the cell viability of each group was observed.

[0042] Colony formation experiment:

[0043] Experimental group: HT-29 and NCM460 cells were suspended and inoculated into 24-well plates at a density of 100 cells per well. After treatment with Statine at a final concentration of 320 μM for 24 h, the medium was replaced with drug-free 1640 complete medium for further incubation for 72 h. Then, the colonies were fixed with 4 wt% paraformaldehyde (4 wt% paraformaldehyde was prepared by dissolving 4 g of paraformaldehyde in 100 ml of PBS solution), and stained with crystal violet to observe the cell colony situation. Western blot was used to detect the protein expression level of HT-29 cells treated with 320 μM of Statine for 24 h, and immunofluorescence detection was performed by 4% paraformaldehyde fixation.

[0044] The control group (CON) was treated with PBS for 24 h, and then replaced with 1640 complete medium without PBS.

[0045] Gene silencing experiments:

[0046] Experimental group 1 (siR-NC): Control RNA (siR-NC) was transfected into HT-29 cells using Lipofectamine 3000. The cells were incubated in 10% CCK8 medium at 37°C in the dark for 1 h, and cell viability was measured using a microplate reader at 450 nm.

[0047] Experimental group 2 (siR-CCL5): CCL5-specific RNA (siR-CCL5) was transfected into HT-29 cells using Lipofectamine 3000. The cells were incubated in 10% CCK8 medium at 37°C in the dark for 1 h, and cell viability was measured using a microplate reader at 450 nm.

[0048] Experimental group 3 (siR-CCL5+STA): CCL5-specific RNA (siR-CCL5) was transfected into HT-29 cells using Lipofectamine 3000. 24 hours after transfection, the cells were treated with 320 μM Statine for 24 hours. Finally, the culture medium was replaced with 10% CCK8 and incubated at 37°C in the dark for 1 hour. Cell viability was measured using a microplate reader at 450 nm.

[0049] Experimental group 4 (STA): HT-29 cells were treated with 320 μM Statine for 24 h, and then the culture medium was replaced with 10% CCK8. The cells were incubated at 37°C in the dark for 1 h, and cell viability was measured using a microplate reader at 450 nm.

[0050] GraphPad Prism 8.0 was used to draw the graphs. The data were expressed as mean ± SEM. One-way ANOVA and Dunnett's post hoc test were used to evaluate the differences among the groups. P < 0.05 was considered to be a significant difference.

[0051] Figure 1Statine inhibits tumor cell growth without affecting normal colon epithelial cell NCM460 activity; wherein a is the cell viability of HT-29, NCM460, MC38 and Caco-2 cells treated with different concentrations of Statine and Mevastatin for 24 h; b is the colony formation ability of HT-29 and NCM460 cells treated with 320 μM of Statine for 24 h; c is the Western blot analysis of protein expression of HT-29 cells treated with 320 μM of Statine for 24 h; d is the immunofluorescence staining result of cleaved caspase-3 of HT-29 cells treated with 320 μM of Statine for 24 h; e is the cell viability of HT-29 cells transfected with siR-NC or siRNA-CCL5 and treated with 320 μM of Statine for 24 h.

[0052] As can be seen from Figure 1 , Statine showed the strongest cytotoxicity in MC38, Caco-2 and HT-29 cells, while it was not toxic to NCM460 cells at the highest concentration; while Mevastatin, although showing stronger cytotoxicity in MC38 and Caco-2, was more toxic to NCM460 and less sensitive to HT-29 cells (as shown in a of Figure 1 ). These findings were further verified by colony formation experiments (as shown in b of Figure 1 ), which showed that Statine significantly inhibited the colony formation ability of HT-29 cells and did not affect the colony formation of NCM460 cells. Western blot analysis further showed that Statine significantly increased the expression of CCL5 and the apoptosis-related protein cleaved caspase-3 (as shown in c of Figure 1 ). The results of immunofluorescence experiments were consistent with this, showing that Statine significantly enhanced the expression of cleaved caspase-3 (as shown in d of Figure 1 ). As shown in e of Figure 1 , inhibition of CCL5 significantly inhibited the effect of STA on reducing the viability of HT-29 cells.

[0053] The above results show that Statine can promote tumor cells to up-regulate CCL5 without affecting the activity of normal colon epithelial cells, thereby inducing apoptosis to occur.

[0054] 2. Tumor inhibition effect of Statine in tumor-bearing mice

[0055] Test method: 4-6 week old male mice were used for the test, and MC38 cells were injected subcutaneously into the right flank of the mice at a dose of 1 × 107 The density of 1 x 106cells / mL was suspended in PBS. After the mice were shaved, 100 μL of cell suspension was injected subcutaneously. The tumor size was measured 10 days after inoculation, and the mice were grouped. The mice in the PBS group were injected intratumorally with 100 μL of PBS, while the mice in the STA group were injected intratumorally with 32 mg / kg of Statine (dissolved in 100 μL of PBS). The tumor size and tumor weight were recorded on day 20. The tumor tissue was collected, fixed with 4% paraformaldehyde, and then paraffin-embedded for hematoxylin-eosin (HE) staining and immunohistochemical detection of proliferation markers (Ki67) and cleaved caspase-3. GraphPad Prism 8.0 was used for plotting, and the data were expressed as mean ± SEM. One-way ANOVA and Dunnett’s post hoc test were used to evaluate the differences between groups, and P < 0.05 was considered to be significantly different.

[0056] Figure 2 Statine inhibited tumor growth in tumor-bearing mice. a, Schematic diagram of tumor-bearing mice modeling and Statine administration; b, Images of MC38 tumors after treatment with STA and PBS; c, Quantitative analysis and counting of MC38 tumor volume after treatment with STA and PBS; d, HE staining analysis of MC38 tumor sections after treatment with STA and PBS; e, Immunohistochemical analysis of Ki67 in MC38 tumors after treatment with STA and PBS; f, Immunohistochemical analysis of cleaved caspase-3 in MC38 tumor tissue after treatment with STA and PBS.

[0057] As shown in FIG. 1, intratumoral injection of Statine in MC38 tumor-bearing mice significantly inhibited the size (b) and weight (c) of the tumors. HE staining of tumor tissue and immunohistochemical analysis of Ki67 and cleaved caspase-3 showed that, compared with intratumoral injection of PBS, intratumoral injection of STA once enhanced the infiltration of immune cells (d), significantly inhibited tumor growth (e), and significantly upregulated the expression of cleaved caspase-3 (f). Figure 2 Figure 2 Figure 2 Figure 2 Figure 2 Figure 2

[0058] The above results show that STA can effectively induce tumor cell apoptosis and inhibit tumor cell growth, and suggest that STA may not only directly act on tumor cells in vivo, but also play an anti-tumor role by recruiting immune cell infiltration.

[0059] ​​​​​​3. Statine inhibits tumor growth in AOM / DSS-induced colorectal tumor model in mice

[0060] Test method: 4-6-week-old male mice were used for the experiment and randomly divided into two groups. Mice in the PBS group were injected intraperitoneally with 10 mg / kg of AOM (azoxymethane) on day 0, and 2wt% DSS (dextran sulfate sodium) was added to the drinking water, as described above. Mice in the Statine group (STA-P) were also treated according to the PBS protocol, but were given 32 mg / kg of Statine through a gastric tube on days 12, 31, and 50, respectively. On day 64, the tumor size, tumor weight, and organ index were recorded. GraphPad Prism 8.0 was used for plotting, and the data were expressed as mean ± SEM. One-way ANOVA and Dunnett's post hoc test were used to evaluate the differences between groups, and P<0.05 was considered to be significantly different.

[0061] Figure 3 Statine inhibits tumor growth in AOM / DSS-induced colorectal tumor model in mice, where a is the mouse colorectal tumor model established by AOM / DSS and STA treatment; b is the mouse colorectal dissection image; c is the quantitative analysis of mouse tumor load; d is the mouse organ index statistical data.

[0062] As shown in Figure 3 , compared with the PBS group, the STA-P group significantly reduced the tumor load of mice ( Figure 3 b and Figure 3 c), and the analysis of multiple organ indices showed that there was no significant difference in kidney, spleen, thymus, and liver between groups ( Figure 3 d).

[0063] The above results show that Statine has the effect of inhibiting tumor growth, and at the same time indicate that the administration of Statine does not have a significant impact on the quality of these organs.

[0064] The above, only for the preferred specific embodiments of the present application, but the scope of protection of the present application is not limited to this, any skilled in the art of the technical personnel in the technical range disclosed by the present application, can easily think of changes or replacement, should be covered within the scope of protection of the present application. Therefore, the scope of protection of the present application should be subject to the scope of protection of the claims.

Claims

1. Application of (3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid in the preparation of anti-colorectal tumor drugs.

2. The use according to claim 1, characterized in that It also includes the use of the pharmaceutically acceptable salt, stereoisomer, solvate, crystal form, isotope label or prodrug of (3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid in the preparation of anti-colorectal tumor drugs.

3. The use according to claim 1 or 2, characterized in that The colorectal tumors include cecal cancer, rectal cancer, colorectal adenocarcinoma and colon cancer.

4. The use according to claim 1 or 2, characterized in that The anti-colorectal tumor drug also includes excipients.

5. The use according to claim 1, characterized in that The (3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid upregulates the expression of chemokine CCL5 in colorectal tumor cells, activates caspase 3 to promote apoptosis of colorectal tumor cells, and thus prevents and / or treats colorectal tumors.

6. An anti-colorectal tumor drug, characterized in that: The (3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid is used as an effective component and is combined with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition.

7. Use of (3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid and an antitumor agent in the preparation of anti-colorectal tumor drugs.

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