Application of geniposide in preparation of antitumor drugs

Abstract

The invention provides an application of geniposide in preparation of antitumor drugs, and finds that the geniposide inhibits the expression and enzymatic activity of TPA-induced matrix metalloproteinase 9 by inducing the expression of heme oxygenase-1 so as to achieve the effect of inhibiting tumor metastasis. A new insight is provided for the molecular mechanism of the geniposide for preventing the development of human tumor cells, which indicates that the geniposide possibly becomes a drug for resisting tumor metastasis, is used for inhibiting human tumors such as breast cancer and the like, and has extremely high scientific research value and practical significance.

Description

Technical Field

[0001] The present invention relates to the application fields of biomedicine and traditional Chinese medicine, and in particular to the application of geniposide in the preparation of anti-tumor drugs. Background Art

[0002] Gardenoside is an iris glycoside found in the fruit of Gardenia jasminoides, which has multiple pharmacological effects, including neuroprotection, antidiabetic, anti-inflammatory and antioxidant activities. Studies have shown that it can protect organisms from oxidative stress by inducing the expression of heme oxygenase-1 (HO-1).

[0003] Currently, researchers have found the beneficial effects of HO-1 induction in blocking tumor migration and invasion in breast cancer cells. Matrix metalloproteinase 9 (MMP-9) is involved in the breakdown of the extracellular matrix during breast cancer metastasis, and drugs that block MMP-9 activation may serve as potential therapeutic candidates for the development of anti-tumor metastasis drugs. 12-O-tetradecanoyl-13-acetate (TPA) is a well-known tumor promoter that can significantly induce the expression and catalytic activity of MMP-9 mRNA in human breast cancer cells. Zinc heme 4 (ZnPP) increased HO-1 gene expression and inhibited TPA-induced MMP-9 enzyme activity and mRNA expression in human breast cancer cells. In the chick embryo chorioallantoic membrane (CAM) experiment, it was found that gardenia glycoside has an anti-angiogenic effect, indicating that gardenia glycoside can be used as a potential adjuvant chemotherapy anti-metastatic drug.

[0004] However, the specific mechanism of HO-1 inducer inhibiting breast tumor metastasis and the anti-angiogenic effect of Gardenia glycoside is still unclear, and the molecular mechanism of Gardenia glycoside in inhibiting breast tumor metastasis is also unclear, which also leads to the lack of clear drug targets for screening inhibiting breast tumor metastasis, and further leads to the lack of safe and effective drugs for inhibiting breast tumor metastasis. Therefore, the existing technology needs to be improved and developed. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an application of Gardenia jasminoides in the preparation of anti-tumor drugs, aiming to solve the problem of the lack of safe and effective drugs for inhibiting breast tumor metastasis in the prior art.

[0006] The technical solution of the present invention is as follows:

[0007] The invention discloses an application of gardenia glycoside in the preparation of anti-tumor drugs.

[0008] In the application, the Gardenia glycoside achieves an anti-tumor effect by inhibiting tumor metastasis and / or anti-angiogenesis.

[0009] In the application, the Gardenia glycoside inhibits the expression and enzyme activity of matrix metalloproteinase 9 by inducing the expression of heme oxygenase-1, thereby achieving the effect of inhibiting tumor metastasis.

[0010] The application, wherein the tumor includes breast cancer.

[0011] In the application, the anti-tumor drug comprises a pharmaceutically acceptable carrier or excipient.

[0012] The application, wherein the anti-tumor drug includes tablets, capsules, injections, oral liquid preparations, and granules.

[0013] Application of geniposide in studying the molecular mechanism of inhibiting tumor metastasis.

[0014] The invention discloses an application of gardenia glycoside in preparing a drug for inhibiting tumor metastasis.

[0015] An application of geniposide in screening candidate drugs for inhibiting tumor metastasis.

[0016] An application of geniposide in screening drug targets for inhibiting tumor metastasis.

[0017] Beneficial effects: The present invention provides an application of gardenia glycoside in the preparation of anti-tumor drugs, and finds that gardenia glycoside inhibits the expression and enzyme activity of matrix metalloproteinase 9 induced by TPA by inducing the expression of heme oxygenase-1, thereby achieving the effect of inhibiting tumor metastasis. The present invention confirms that gardenia glycoside is a newly discovered heme oxygenase-1 inducer and its anti-tumor effect, which provides new insights into the molecular mechanism of gardenia glycoside in preventing the progression of human tumor cells, indicating that gardenia glycoside may become an anti-tumor metastasis drug and be used to inhibit human tumors such as breast cancer and other diseases, which has extremely high scientific research value and practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the results of MMP-9 mRNA expression levels in cells after treatment with geniposide provided in an embodiment of the present invention.

[0019] Figure 2 This is a schematic diagram of the MMP-9 enzyme activity results of cells after being treated with geniposide provided in an embodiment of the present invention.

[0020] Figure 3 This is a schematic diagram of the HO-1 mRNA expression level results of cells after geniposide treatment provided in an embodiment of the present invention.

[0021] Figure 4 This is a schematic diagram of the HO-1 protein expression level results of cells after geniposide treatment provided in an embodiment of the present invention.

[0022] Figure 5 This is a schematic diagram of the results of MMP-9 mRNA expression levels of cells after treatment with different drugs provided in an embodiment of the present invention.

[0023] Figure 6 This is a schematic diagram of the MMP-9 enzyme activity results of cells after being treated with different drugs provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0024] The present invention provides an application of geniposide in the preparation of an anti-tumor drug. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] The embodiment of the present invention provides an application of geniposide in the preparation of an anti-tumor drug.

[0026] In some embodiments, the Gardenia jasminoides glycoside achieves an anti-tumor effect by inhibiting tumor metastasis and / or anti-angiogenesis.

[0027] In some embodiments, the Gardenia glycoside inhibits the expression and enzyme activity of matrix metalloproteinase 9 (MMP-9) by inducing the expression of heme oxygenase-1 (HO-1), thereby achieving the effect of inhibiting tumor metastasis.

[0028] In some embodiments, the geniposide inhibits the expression and enzyme activity of MMP-9 induced by 12-O-tetradecanoyl-13-acetate (TPA) by inducing the expression of HO-1.

[0029] In some embodiments, the tumor comprises breast cancer.

[0030] In a specific embodiment, the present invention provides an application of geniposide in the preparation of an anti-breast cancer metastasis drug. The geniposide inhibits the expression and enzyme activity of MMP-9 induced by TPA by inducing the expression of HO-1, thereby achieving the effect of anti-breast cancer metastasis.

[0031] Matrix metalloproteinases (MMPs) are key regulators of extracellular matrix and basement membrane degradation. Overexpression of matrix metalloproteinases promotes tumor invasion, migration and angiogenesis in a variety of cancer cell lines. In particular, matrix metalloproteinase 9 (MMP-9) can specifically degrade type IV collagen in breast cancer cells, thereby promoting tumor metastasis. In order to determine whether the inhibitory effect of gardenia glycosides on blood vessels is related to the expression of MMP-9 induced by 12-O-tetradecanoyl-13-acetate (TPA), the present invention detected the mRNA level of MMP-9 and the enzyme activity of MMP-9 in breast cancer cells treated with gardenia glycosides. The results showed that TPA significantly induced the expression of MMP-9 mRNA and the activity of MMP-9 in breast cancer cell lines MCF-7 cells and MDA-MB-231 cells. Gardenia glycosides can inhibit the induction of MMP-9 mRNA expression by TPA in a dose-dependent manner. Gardenia glycosides also reduce the enzyme activity of MMP-9 induced by TPA.

[0032] HO-1 has been identified as a tumor suppressor due to its inhibitory activity on tumor metastasis. The present invention found that geniposide dose-dependently induced the expression of HO-1 mRNA and HO-1 protein. In addition, the present invention also verified the role of HO-1 in MMP-9 expression and enzyme activity by using the HO-1 inhibitor ZnPP. ZnPP treatment significantly rescued the inhibitory effect of geniposide on TPA-induced MMP-9 expression and enzyme activity in MDA-MB-231 cells and MCF-7 cells.

[0033] The present invention found that gardenia glycosides inhibited the expression and enzyme activity of MMP-9 mRNA induced by TPA by inducing the expression of HO-1, thereby achieving the effect of inhibiting tumor metastasis. This provides new insights into the molecular mechanism of gardenia glycosides in preventing the progression of human tumor cells, indicating that gardenia glycosides may become a drug for anti-tumor metastasis and be used to inhibit human tumors such as breast cancer. Based on this, the present invention provides the use of gardenia glycosides in the preparation of anti-tumor drugs.

[0034] In some embodiments, the anti-tumor drug includes a pharmaceutically acceptable carrier or excipient.

[0035] In some embodiments, the anti-tumor drug includes tablets, capsules, injections, oral liquid preparations, and granules.

[0036] Gardenia glycoside is a known substance with pharmaceutical activity. Gardenia glycoside can be prepared into any clinically applicable pharmaceutical preparation, such as powder, tablet, pellet, capsule, microcapsule, injection, granule or liquid, etc., according to conventional preparation methods.

[0037] In the process of preparing the above preparations, Gardenia jasminoides can be used alone, or can be mixed with any excipient suitable for preparing clinical medicaments to prepare pharmaceutical preparations.

[0038] The embodiment of the present invention also provides an application of geniposide in studying the molecular mechanism of inhibiting tumor metastasis.

[0039] The embodiment of the present invention also provides an application of Gardenia jasminoides in preparing a drug for inhibiting tumor metastasis.

[0040] The embodiment of the present invention also provides an application of Gardenia jasminoides in screening candidate drugs for inhibiting tumor metastasis.

[0041] The embodiment of the present invention also provides an application of Gardenia jasminoides in screening drug targets for inhibiting tumor metastasis.

[0042] The application of the present invention in preparing anti-tumor drugs is further explained by using specific examples below:

[0043] Unless otherwise specified, all reagents and materials used in the examples are commercially available.

[0044] Example 1 Human breast cancer cell line culture

[0045] Human breast cancer cell lines MDA-MB-231 and MCF-7 were cultured in cell culture dishes in a modified Eagle medium (pH 7.2) supplemented with 1.5 g / L NaHCO 3 , 10% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin in a humidified incubator at 37° C. and 5% CO 2 .

[0046] Example 2 Gardenia glycoside inhibits TPA-induced MMP-9 mRNA expression and enzyme activity

[0047] The cells MDA-MB-231 and MCF-7 cultured in Example 1 were pretreated with 0-60 μM concentration gradient of geniposide for 24 hours, and then stimulated with 50 ng / ml TPA for 24 hours. The expression level of MMP-9 mRNA and MMP-9 enzyme activity of the cells were then detected.

[0048] 1. Detection of MMP-9 mRNA expression level in cells

[0049] Total mRNA of cells treated with different concentrations of Gardenia jasminoides was collected, and the expression of MMP-9 mRNA was detected by real-time fluorescence quantitative PCR. The real-time RT-PCR conditions were as follows: the PCR mixture system was a 10 μL reaction system, including 1 μL cDNA template, 0.5 μL forward primer (10 μM) and 0.5 μL reverse primer (10 μM), 5 μL 2×SYBR Green PCR master mix, and the volume was made up with double distilled water.

[0050] The primer sequences are as follows:

[0051] SEQ.ID NO 1:MMP-9-F,5′-GAACCAATCTCACCGACAGG-3′

[0052] SEQ.ID NO 2:MMP-9-R,5′-GCCACCCGAGTGTAACCATA-3′

[0053] Real-time fluorescence quantitative PCR results Figure 1 As shown, Figure A is the cell line MDA-MB-231, and Figure B is the cell line MCF-7. The results are expressed as mean ± standard deviation (n = 3). The results were statistically analyzed using a one-way analysis of variance test. # represents a comparison with the control group, P < 0.05. * represents a comparison with the TPA treatment group, P < 0.05. As can be seen from the figure, Gardenia glycoside inhibited the expression level of MMP-9 mRNA induced by TPA in a dose-dependent manner.

[0054] 2. Detection of MMP-9 enzyme activity in cells

[0055] Gelatin zymography (Zym) was used to detect the enzyme activity of MMP-9 in breast cancer cells. The cells treated with geniposide were washed with PBS and treated with different concentrations of 12-O-tetradecanoyl-13-acetate (TPA) and geniposide in serum-free medium for 24 hours. 1 mg / mL gelatin was used as the substrate, and 20 μL of the supernatant was electrophoresed using a 10% SDS-polyacrylamide gel. After electrophoresis, the gel was rinsed with 2.5% Triton X-100 solution to remove SDS, and then incubated in a refolding buffer (50 mM Tris, 10 mM CaCl2, 1 μM ZnCl2, 200 mM sodium chloride, pH 7.4) at 37°C overnight. Then it was stained with 0.1% Coomassie Brilliant Blue R-250 and destained with acetic acid. The unstained area of ​​the gel is MMP-9.

[0056] The results are as follows Figure 2As shown, Figure A is the cell line MDA-MB-231, and Figure B is the cell line MCF-7. As can be seen from the figure, Gardenia glycoside inhibits TPA-induced MMP-9 enzyme activity in a dose-dependent manner.

[0057] comprehensive Figure 1-2 Based on the results, it can be concluded that Gardenia glycoside can inhibit TPA-induced MMP-9 mRNA expression and enzyme activity in a dose-dependent manner.

[0058] Example 3 Gardenia glycoside induces HO-1 mRNA and protein expression

[0059] The cells MDA-MB-231 and MCF-7 cultured in Example 1 were pretreated with 0-60 μM geniposide for 24 hours, and then stimulated with 50 ng / ml TPA for 24 hours. The expression levels of HO-1 mRNA and protein in the cells were then detected.

[0060] 1. Real-time fluorescence quantitative PCR detection of HO-1 mRNA expression level in cells

[0061] The detection method and conditions are the same as in Example 2, wherein the primer sequences are as follows:

[0062] SEQ.ID NO 3:HO-1-F,5′-GGGTGATAGAAGAGGCCAAGA-3′

[0063] SEQ.ID NO 4:HO-1-R,5′-AGCTCCTGCAACTCCTCAAA-3′

[0064] Real-time fluorescence quantitative PCR results Figure 3 As shown, Figure A is the cell line MDA-MB-231, and Figure B is the cell line MCF-7. The results are expressed as mean ± standard deviation (n = 3). The results were statistically analyzed using a one-way analysis of variance test. * represents P < 0.05. As can be seen from the figure, Gardenia glycoside induced the expression of HO-1 mRNA in a dose-dependent manner under TPA induction.

[0065] 2. Western blot analysis of HO-1 protein expression levels in cells

[0066] Total cell proteins treated with different concentrations of Gardenia jasminoides were collected for immunoblotting experiments. The obtained proteins were separated by 12% SDS polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membrane (immobilon-P membrane). After blocking the blot in 2% bovine serum albumin solution, the membrane was incubated with primary antibodies against HO-1 and β-actin overnight, and then incubated with secondary antibodies conjugated to horseradish peroxidase for 1 hour. Specific bands were detected using a fixed chemiluminescent detection reagent (Millipore) and photographed using a gel Doc imager. The results were analyzed using one-way analysis of variance (GraphPad software) to determine statistical significance. The results are expressed as mean ± SEM. When the P value <0.05, its significance was accepted.

[0067] The results are as follows Figure 4 As shown, Figure A is the cell line MDA-MB-231, and Figure B is the cell line MCF-7. As can be seen from the figure, Gardenia glycoside induces HO-1 protein expression in a dose-dependent manner under TPA induction.

[0068] comprehensive Figure 3-4 Based on the results, it can be concluded that Gardenia glycoside induces HO-1 mRNA and protein expression in a dose-dependent manner under TPA induction.

[0069] Example 4 ZnPP alleviates the inhibitory effect of geniposide on TPA-induced MMP-9 expression and enzyme activity

[0070] First, the cells MDA-MB-231 and MCF-7 cultured in Example 1 were pretreated under the following pretreatment conditions: (1) cells were pretreated with 60 μM gardenia glycoside and 10 μM ZnPP for 24 hours, and then treated with 50 ng / ml TPA for another 48 hours; (2) cells were pretreated with 60 μM gardenia glycoside for 24 hours, and then treated with 50 ng / ml TPA for another 48 hours; (3) cells were pretreated without gardenia glycoside for 24 hours, and then treated with 50 ng / ml TPA for another 48 hours; (4) control group, no drug was added during pretreatment.

[0071] The expression level of MMP-9 mRNA and enzyme activity of cells after different treatments were detected respectively, and the detection method, primers, etc. were the same as those in Example 2. The results are shown in FIG. Figure 5 (MMP-9 mRNA expression level) and Figure 6 (MMP-9 enzyme activity) is shown in Figure A, where Figure A is the cell line MDA-MB-231 and Figure B is the cell line MCF-7. As can be seen from the figure, the HO-1 inhibitor ZnPP alleviated the inhibitory effect of Gardenia glycoside on TPA-induced MMP-9 expression and enzyme activity.

[0072] In summary, the present invention provides an application of gardenia glycoside in the preparation of anti-tumor drugs, and it is found that in breast cancer cell lines MDA-MB-231 and MCF-7, gardenia glycoside inhibits the expression and enzyme activity of MMP-9 mRNA induced by TPA by inducing the expression of heme oxygenase-1 (HO-1), thereby achieving the effect of inhibiting tumor metastasis. The present invention confirms that gardenia glycoside is a newly discovered heme oxygenase-1 inducer and its anti-tumor effect, which provides new insights into the molecular mechanism of gardenia glycoside in preventing the progression of human tumor cells, indicating that gardenia glycoside may become an anti-tumor metastasis drug and be used to inhibit human tumors such as breast cancer and other diseases, which has extremely high scientific research value and practical significance.

[0073] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An application of Gardenia jasminoides in the preparation of anti-tumor drugs.

2. The use according to claim 1, characterized in that: The gardenia glycoside achieves an anti-tumor effect by inhibiting tumor metastasis and / or resisting angiogenesis.

3. The use according to claim 2, characterized in that: The gardenia glycoside inhibits the expression and enzyme activity of matrix metalloproteinase 9 by inducing the expression of heme oxygenase-1, thereby achieving the effect of inhibiting tumor metastasis.

4. The use according to claim 1, characterized in that: The tumor includes breast cancer.

5. The use according to claim 1, characterized in that: The anti-tumor drug includes a pharmaceutically acceptable carrier or excipient.

6. The use according to claim 1, characterized in that: The anti-tumor drugs include tablets, capsules, injections, oral liquid preparations, and granules.

7. An application of Gardenia jasminoides in studying the molecular mechanism of inhibiting tumor metastasis.

8. Use of Gardenia jasminoides in the preparation of drugs for inhibiting tumor metastasis.

9. An application of Gardenia jasminoides in screening candidate drugs for inhibiting tumor metastasis.

10. An application of Gardenia jasminoides in screening drug targets for inhibiting tumor metastasis.

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