Application of notoginseng saponins R1 in diagnosis and treatment of ovarian cancer

Abstract

The application discloses application of panax notoginseng saponin R1 in diagnosis and treatment of ovarian cancer and belongs to the field of medical biology.The first object of the application is to disclose application of a natural traditional Chinese medicine component in preparation of a medicine for treating ovarian cancer, and the panax notoginseng saponin R1 belongs to the technical field of new purposes of medicines.The application finds that the panax notoginseng saponin R1 has a significant inhibitory effect on the growth of human ovarian cancer cell strains A2780 and SK-OV-3 through in-vitro anti-tumor activity evaluation, and simultaneously significantly induces cell apoptosis.In addition, the other object also provides a molecular action mechanism and a target point of the panax notoginseng saponin R1 for inhibiting ribosome synthesis; the in-vitro anti-tumor evaluation finds that the natural compound panax notoginseng saponin R1 significantly inhibits proliferation, invasion and migration of ovarian cancer cells and promotes apoptosis of the ovarian cancer cells, and experiments prove that the panax notoginseng saponin R1 inhibits ribosome synthesis by inhibiting activation of an mTOR-RPS6 signal path, and then plays an anti-tumor role.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical biotechnology, and particularly relates to the application of notoginsenoside R1 in diagnosis and treatment of ovarian cancer. BACKGROUND

[0002] Cancer is one of the leading causes of death in the world. Modern targeted drug therapy greatly improves the prognosis of cancer patients. However, advanced metastatic cancer is still incurable. Therefore, it is still necessary to find safer and more effective prevention and treatment methods to improve the treatment effect of cancer and reduce the treatment cost. In the past few years, the use of natural plant preparations for cancer prevention has attracted attention (references 4, 5), and various phytochemicals and plant preparations have been proven to have the function of killing cancer cells by inducing apoptosis (reference 6). Therefore, natural compounds have broad application prospects in the research and development of antitumor drugs.

[0003] Cancer cells must maintain a high rate of ribosome biosynthesis in order to grow and proliferate rapidly. More and more studies have shown that abnormal ribosome biosynthesis leads to the occurrence and development of tumors. Exploring effective targeted drugs for tumors against ribosome synthesis has been a research hotspot in recent years. Several clinically approved anticancer drugs can inhibit ribosomal RNA biosynthesis, but there are few studies on selective drugs targeting ribosome synthesis, mostly due to the lack of discovery and identification of specific related ribosome synthesis targets.

[0004] Ribosome biosynthesis is an important biological process in cells, which directly regulates the rate of protein synthesis in cells. Cancer cells need to increase protein synthesis to maintain uncontrolled proliferation, which in turn leads to abnormal regulation of ribosome synthesis. Studies have shown that abnormal ribosome biosynthesis also promotes the occurrence and development of tumors, so ribosome synthesis is crucial to tumors. Targeting genes related to ribosome synthesis is an effective cancer treatment strategy. Studies have shown that actinomycin D prevents RNA polymerase transcription at the replication fork by inserting ribosomal DNA (reference 1); oxaliplatin can cause ribosome synthesis defects to kill lymphoma cells (reference 2). CX-3543, a small molecule nucleolus targeting agent, is the first G-quadruplex interacting agent to enter human clinical trials. It accumulates in the nucleolus of cancer cells, causing nucleolar protein redistribution, selectively disrupting the nucleolin / rDNA G-quadruplex complex in the nucleolus, thereby inhibiting RNA polymerase I transcription and inhibiting ribosomal RNA synthesis and inducing apoptosis in cancer cells (reference 3). Therefore, the research and development of ribosome synthesis targeted drugs and the elucidation of their molecular mechanisms are of great significance to the treatment of cancer patients.

[0005] As a traditional Chinese medicine, modern pharmacological studies have shown that Panax notoginseng contains saponins, polysaccharides, and flavonoids. Notoginsenoside R1 (NGR1) is the main component of Panax notoginseng, which has various pharmacological effects, including prevention and treatment of cardiovascular diseases, immune regulation, neuroprotection, anti-inflammatory, and anti-tumor. Current studies have shown that NGR1 has significant anti-cancer effects on breast cancer, colorectal cancer, and osteosarcoma. However, there is no report on the application of NGR1 in ovarian cancer, such as its inhibition of ribosome synthesis and its anti-tumor effects. SUMMARY

[0006] To overcome the shortcomings of the prior art, the present application provides the application of notoginsenoside R1 in the diagnosis and treatment of ovarian cancer. High-throughput screening of ribosome synthesis inhibitors is performed using a high-content cell imaging system, and it is found that notoginsenoside R1 can significantly inhibit ribosome synthesis.

[0007] More specifically, the present application has three main technical ideas:

[0008] Firstly, the first object of the present application is to disclose the application of a natural Chinese medicine component in the preparation of a drug for treating ovarian cancer, notoginsenoside R1, which belongs to the technical field of new uses of drugs. Through in vitro anti-tumor activity evaluation, it is found that notoginsenoside R1 has a significant inhibitory effect on the growth of human ovarian cancer cell lines A2780 and SK-OV-3, and can significantly induce cell apoptosis.

[0009] Secondly, the present application also provides the molecular mechanism and target of notoginsenoside R1 in inhibiting ribosome synthesis.

[0010] More specifically, the present application provides a screening method for an inhibitor that can effectively inhibit ribosome synthesis in ovarian cancer, which includes the following steps:

[0011] Step one: label the cell nucleus DNA with EdU and the ribosome RNA with EU, and use a high-content cell imaging analysis system to screen a kinase inhibitor library, obtaining an inhibitor that can simultaneously reduce the EdU and EU fluorescence signals as a candidate drug;

[0012] Step two: analyze and quantify the high-content imaging data to select a candidate drug that can simultaneously inhibit ribosome generation and cell proliferation in ovarian cancer cells;

[0013] Step three: use MTT cell proliferation experiments to detect the effects of different concentration gradients of the candidate drug on the proliferation of ovarian cancer cells;

[0014] Step four: use flow cytometry analysis to detect the effects of different concentration gradients of the candidate drug on the apoptosis of ovarian cancer cells;

[0015] Step five: Transwell migration and invasion experiment is used to detect the influence of different concentration gradients of the candidate drug on the migration and invasion of the ovarian cancer cells.

[0016] As a further optimization of the first aspect, the candidate drug is notoginsenoside R1.

[0017] More specifically, the second aspect of the present application provides an application of the RPS6 phosphorylated protein as a target material for the notoginsenoside R1 in the ovarian cancer.

[0018] More specifically, the third aspect of the present application provides an application of the inhibitor capable of simultaneously reducing the EU and EdU fluorescent signals in the preparation of the ovarian cancer diagnosis reagent.

[0019] As a further optimization of the third aspect method, the inhibitor inhibits the ribosome synthesis in the ovarian cancer cells by controlling the mTOR / RPS6 / 4EBP1 signal pathway activation.

[0020] Further, the inhibitor is notoginsenoside R1.

[0021] More specifically, the fourth aspect of the present application provides an application of the inhibitor capable of simultaneously reducing the EU and EdU fluorescent signals in the preparation of the ovarian cancer diagnosis kit.

[0022] As a further optimization of the fourth aspect method, the inhibitor inhibits the ribosome synthesis in the ovarian cancer cells by controlling the mTOR / RPS6 / 4EBP1 signal pathway activation.

[0023] Further, the inhibitor is notoginsenoside R1.

[0024] More specifically, the fifth aspect of the present application provides an application of the notoginsenoside R1 in the preparation of the ovarian cancer antitumor drug, characterized in that the notoginsenoside R1 promotes the apoptosis and inhibits the proliferation of the ovarian cancer cells by inhibiting the ribosome synthesis in the ovarian cancer cells through the control of the mTOR / RPS6 / 4EBP1 signal pathway activation.

[0025] In summary, the present application mainly has the following beneficial effects:

[0026] The in vitro antitumor evaluation finds that the natural compound notoginsenoside R1 significantly inhibits the proliferation, invasion and migration of the ovarian cancer cells and promotes the apoptosis of the ovarian cancer cells, and the experiment proves that the notoginsenoside R1 inhibits the ribosome synthesis by inhibiting the mTOR-RPS6 signal pathway activation, thereby playing an antitumor role. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1(A, B) Panax notoginseng saponin R1 inhibited the generation of ribosomal RNA in the nucleus of ovarian cancer A2780 cells; (C, D) Panax notoginseng saponin R1 inhibited the proliferation of ovarian cancer cells A2780. *P < 0.05, **P < 0.01.

[0028] Figure 2 Panax notoginseng saponin R1 inhibited the proliferation of ovarian cancer cells (**P < 0.01, ***P < 0.001); (A) ovarian cancer A2780 cells were treated with different concentrations of Panax notoginseng saponin R1; (B) ovarian cancer SK-OV-3 cells were treated with different concentrations of Panax notoginseng saponin R1.

[0029] Figure 3 Panax notoginseng saponin R1 promoted the apoptosis of ovarian cancer cells.

[0030] Figure 4 Panax notoginseng saponin R1 inhibited the migration and invasion of ovarian cancer cells.

[0031] Figure 5 Panax notoginseng saponin R1 inhibited the synthesis of ribosomes in tumor cells.

[0032] Figure 6 Panax notoginseng saponin R1 inhibited the synthesis of ribosomes in ovarian cancer by regulating the mTOR / RPS6 / 4EBP1 signaling pathway. (A) Western blotting was used to detect the proteins or phosphorylated proteins shown in the figure in ovarian cancer cell lines; (B) quantification data of the expression levels of phosphorylated proteins. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0034] The present application provides a natural compound Panax notoginseng saponin R1 for inhibiting the synthesis of ribosomes in ovarian cancer, and regulates the mTOR / RPS6 / 4EBP1 signaling pathway. The specific screening method is as follows:

[0035] (1) Use EdU to label cell nucleus DNA, EU to label ribosomal RNA, use high-content cell imaging analysis system, screen kinase inhibitor library, and the inhibitor that can simultaneously reduce EdU and EU fluorescence signal is the candidate drug of the present application.

[0036] (2) analyze and quantify high-content imaging data, and the natural compound candidate drug Panax notoginseng saponin R1 can simultaneously inhibit the ribosome generation and cell proliferation of ovarian cancer cells.

[0037] (3) MTT cell proliferation assay was used to detect the effect of different concentrations of Panax notoginseng saponins Rl on the proliferation of ovarian cancer cells;

[0038] (4) Flow cytometry was used to detect the effect of different concentrations of Panax notoginseng saponins Rl on the apoptosis of ovarian cancer cells;

[0039] (5) Transwell migration and invasion assay was used to detect the effect of different concentrations of Panax notoginseng saponins Rl on the migration and invasion of ovarian cancer cells.

[0040] Example 1: Drug screening

[0041] 1. The screening process is as follows:

[0042] Human ovarian cancer A2780 cells and SK-OV-3 cells in logarithmic growth phase were plated in 96-well plates, and 72 different kinase inhibitors were added after the cells adhered, with each three wells as a repeated treatment. After 48 hours of culture, 5-ethynyluridine (5-ethynyluridine, EU) was used to detect the synthesis of new RNA in cells, and EdU (5-ethynyl-2'-deoxyuridine) was used to detect the synthesis of new DNA to detect cell proliferation. DAPI was used to stain the cell nucleus, and after staining, high-content microscopic imaging system was used for imaging and analysis. The nuclei of labeled living cells showed blue fluorescence, the newly formed nucleolar RNA labeled with EU showed green fluorescence, and the cells in the proliferation state were labeled with EdU and showed bright red fluorescence. Image J software was used to quantify the fluorescence signal intensity, and the integral optical density value obtained was divided by the number of cells in the field identified by DAPI staining to obtain the quantitative value of EU. The number of EdU positive cells was calculated, and the EdU positive cell rate = EdU positive cell number / total cell number in the same field x 100%. Compared with the control solvent DMSO treatment, the inhibitors that reduce EU and EdU fluorescence signals are candidate drugs.

[0043] 2. Natural compound Panax notoginseng saponins Rl is listed as a candidate drug

[0044] The results are shown in Figure 1 Compared with the control group, the Panax notoginseng saponins Rl treatment group significantly reduced the fluorescence intensity of EU in the nuclei of A2780 cells, i.e. inhibited the generation of ribosomal RNA in the nuclei of A2780 cells; at the same time, the number of EdU positive cells in A2780 cells was reduced, i.e. the proliferation of A2780 cells was inhibited.

[0045] Example 2: Panax notoginseng saponins Rl inhibits ovarian cancer cell proliferation

[0046] Ovarian cancer A2780, SK-OV-3 cells were treated with different concentrations of Panax notoginseng saponins R1 and DMSO as control. After 72 hours of treatment, MTT was used to detect cell proliferation. The results are shown in Figure 2 Panax notoginseng saponins R1 significantly inhibited the proliferation of A2780 cells and SK-OV-3 cells in a concentration gradient-dependent manner.

[0047] Example 3: Panax notoginseng saponins R1 promotes ovarian cancer cell apoptosis

[0048] After 24 hours of treatment with different concentrations of Panax notoginseng saponins R1 75 mmol / L and 150 mmol / L in A2780 cell lines and SK-OV-3 cell lines, respectively, flow cytometry was used to analyze the effect of Panax notoginseng saponins R1 on ovarian cancer cell apoptosis. The results are shown in Figure 3 Compared with the control group, Panax notoginseng saponins R1 promoted the apoptosis of A2780 cells and SK-OV-3 cells in a concentration gradient-dependent manner (p<0.05).

[0049] Example 4: Panax notoginseng saponins R1 inhibits ovarian cancer cell migration and invasion

[0050] After treatment with different concentrations of Panax notoginseng saponins R1 75 mmol / L and 150 mmol / L in SK-OV-3 cell lines, respectively, Transwell migration and invasion experiments were used to analyze the effect of Panax notoginseng saponins R1 on ovarian cancer cell migration and invasion. The results are shown in Figure 4 Compared with the control group, Panax notoginseng saponins R1 inhibited the migration and invasion of SK-OV-3 cells in a concentration gradient-dependent manner (p<0.001).

[0051] Example 5: Panax notoginseng saponins R1 inhibits ribosome synthesis in ovarian cancer cells

[0052] After treatment of ovarian cancer cells A2780 and SK-OV-3 with Panax notoginseng saponins R1, total RNA was extracted, and the expression of ribosomal RNA genes and ribosome synthesis-related transcription factors in the cells was detected by fluorescent quantitative PCR. These genes include ribosomal RNA 5.8S rRNA, 18S rRNA, 28S rRNA, Pre-45S rRNA, 18S rRNA 5ˊETS, and transcription factors RRN3 and NCL. The results are shown in Figure 5 After treatment with Panax notoginseng saponins R1, the expression of ribosomal RNA (5.8S rRNA, 18S rRNA, 28S rRNA, Pre-45S rRNA, 5ˊETS) and transcription factors RRN3 and NCL in ovarian cancer cells was significantly inhibited (p<0.05).

[0053] (A, B) Panax notoginseng saponin R1 inhibits the expression of ribosomal RNA and important transcription factors in the ribosome synthesis pathway in the nuclei of ovarian cancer A2780 cells; (C, D) Panax notoginseng saponin R1 inhibits the expression of ribosomal RNA and important transcription factors in the ribosome synthesis pathway in the nuclei of ovarian cancer SK-OV-3 cells.

[0054] Example 6: Panax notoginseng saponin R1 inhibits ribosome synthesis in ovarian cancer cells by inhibiting the activation of mTOR / RPS6 signaling pathway

[0055] p-RPS6 is the phosphorylated activated form of RPS6, which is a key molecule downstream of the mTOR signaling pathway, and a large number of studies have shown that the mTOR signaling pathway plays a crucial role in ribosome synthesis. The present application detects the expression of p-mTOR, mTOR, p-RPS6, RPS6, p-4EBP1 and 4EBP1 proteins in ovarian cancer cells by Western Blot, and the results are shown in 6. Compared with the control group, the expression of p-mTOR, p-RPS6 and p-4EBP1 proteins in the tumor of the Panax notoginseng saponin R1 treatment group was significantly inhibited. It is shown that in ovarian cancer cell lines A2780 and SK-OV-3, Panax notoginseng saponin R1 significantly inhibits the expression of p-RPS6 and p-4EBP1 proteins.

[0056] References

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[0058] 2. Bruno PM, Liu Y, Park GY. A subset of platinum-containing chemotherapeutic agents kills cells by inducing ribosome biogenesis stress. Nat Med, 2017, 23: 461-471.

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[0060] 4. Kumar M, Kaur V, Kumar S, et al. Phytoconstituents as apoptosis inducing agents: strategy to combat cancer. Cytotechnology, 2016, 68(4):531-563.

[0061] 5. Manson MM. Cancer prevention-the potential for diet to modulate molecular signalling. Trends Mol Med, 2003, 9(1):8-11.

[0062] 6. Bayala B, Zoure AA, Baron S, et al. Pharmacological Modulation of Steroid Activity in Hormone-Dependent Breast and Prostate Cancers: Effect of Some Plant Extract Derivatives. Int J Mol Sci, 2020, 21(10):3690.

[0063] While embodiments of the present application have been shown and described herein, it will be understood by those of ordinary skill in the art that various changes, modifications, alternatives, and variations can be made to these embodiments without departing from the principles and spirit of the application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of Panax notoginseng saponin R1 in the preparation of drugs for treating ovarian cancer, characterized in that: The notoginsenoside R1 promotes apoptosis and inhibits proliferation of ovarian cancer cells by controlling the activation of the mTOR / RPS6 / 4EBP1 signaling pathway and inhibiting ribosome synthesis in ovarian cancer cells.

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