阅读说明：本技术 乌骨藤提取物c21甾体在制备促进胃癌细胞凋亡药物中的应用 (Application of glaucescent fissistigma root extract C21 steroid in preparation of medicine for promoting gastric cancer cell apoptosis ) 是由 张威 王震 应优敏 李凯强 张昱 陈以栗 郝珂 于 2020-02-25 设计创作，主要内容包括：本发明公开了一种C21甾体在制备促进癌细胞凋亡药物中的应用,所述C21甾体通过抑制自噬促进细胞凋亡而抑制细胞的增殖。还公开了一种C21甾体在制备治疗胃癌药物中的应用,所述C21甾体为乌骨藤提取物,所述C21甾体通过抑制自噬促进凋亡而抑制胃癌细胞增殖。本发明公开的C21甾体可显著抑制癌细胞生长和促进细胞凋亡的发生,为揭示C21甾体抗胃癌活性的新机制提供理论基础。(The invention discloses application of a C21 steroid in preparation of a medicine for promoting cancer cell apoptosis, wherein the C21 steroid inhibits cell proliferation by inhibiting autophagy to promote cell apoptosis. The C21 steroid is a glaucescent fissistigma root extract, and the C21 steroid inhibits gastric cancer cell proliferation by inhibiting autophagy and promoting apoptosis. The C21 steroid disclosed by the invention can obviously inhibit the growth of cancer cells and promote the occurrence of apoptosis, and provides a theoretical basis for disclosing a new mechanism of the anti-gastric cancer activity of the C21 steroid.)

1. The use of a C21 steroid for the preparation of a medicament for promoting apoptosis in cancer cells, wherein the C21 steroid inhibits proliferation of cancer cells by inhibiting autophagy to promote apoptosis.

2. The use of the C21 steroid for the preparation of a medicament for promoting apoptosis in cancer cells, wherein the C21 steroid promotes the development of the autophagy pathway by activating AKT.

3. The use of the C21 steroid of claim 1, wherein the C21 steroid is cochinchina sylvine extract for the preparation of a medicament for promoting apoptosis.

4. The use of the C21 steroid for the preparation of a medicament for promoting apoptosis in a human of claim 1, wherein the cancer cell is a gastric cancer cell.

5. The application of the C21 steroid in preparing a medicine for treating gastric cancer is characterized in that the C21 steroid is a glaucescent fissistigma root extract, and the C21 steroid inhibits proliferation of gastric cancer cells by inhibiting autophagy and promoting apoptosis.

Technical Field

The invention relates to the technical field of biology, in particular to application of a glaucescent fissistigma root extract C21 steroid in preparation of a medicine for promoting gastric cancer cell apoptosis.

Background

Gastric cancer is the third most common cancer worldwide and is one of the leading causes of cancer-related deaths. The lack of effective treatment results in short life cycle and poor quality of life for cancer patients. At present, gastric cancer patients are treated mainly by surgical resection and chemotherapy, but the treatment effect is limited. Therefore, there is an urgent need to develop new treatment regimens to improve patient prognosis.

Autophagy is a conserved eukaryotic catabolic reaction that catabolizes cytoplasmic proteins and organelles. As a cytoprotective process, it helps maintain the homeostasis of cancer cells and serves as an alternative energy source in harsh environments. This suggests that autophagy may have a protective role in the development of tumors. Recent studies have shown that key autophagy genes, including autophagy-related genes (ATGs), can cause cellular ROS damage through cell death. Cully et. found that modulating the expression of the cancer suppressor genes PTEN and p53 could positively modulate autophagy. It is well known that oncogene products such as BCL2 anti-apoptotic proteins interact with evolutionarily conserved autophagy proteins, and that binding to Beclin-1 may help maintain autophagy at a level compatible with cell survival. With respect to gastric cancer, recent studies have shown that autophagy has a dual effect on the disease. Many studies have shown that the anticancer drug akebia saponin PA promotes apoptosis of AGS cells by activating autophagy and apoptosis. In contrast, another study reported that the use of an autophagy inhibitor enhanced the cytotoxicity of an anticancer drug in the gastric cancer cell line SGC-7901. Thus, autophagy may be a potential therapeutic target for the treatment of gastric cancer.

Marsdenia Tenacissima (Marsdenia Tenacissima) is a Fissistigma cucurbitae plant which is widely produced in China, and a liquid extract (trade name: Xiaoaiping) thereof is approved to be used for treating esophageal cancer, lung cancer, leukemia, liver cancer and gastric cancer. It has been found that MTE is rich in C21 steroidal glycosides, and that treatment of FR5 fraction can induce apoptosis through PTEN-AKT-mTOR signaling pathway. However, the potential efficacy of C21 steroids on autophagy and whether inhibition of autophagy could promote the anticancer effects of C21 have not been investigated.

Therefore, the present invention mainly examines whether the C21 steroid can induce autophagy and whether autophagy inhibition contributes to the anticancer effect of the C21 extract, and also explores the potential molecular mechanism of inducing autophagy.

Disclosure of Invention

The invention aims to provide application of a C21 steroid in preparation of a medicine for enhancing cancer cell apoptosis, so as to solve the problems in the prior art, and the C21 steroid can obviously inhibit the growth of cancer cells and promote apoptosis so as to inhibit cell proliferation.

The invention also aims to provide application of the C21 steroid in preparation of a medicine for treating gastric cancer, wherein the C21 steroid has the effects of inducing apoptosis and autophagy increase of gastric cancer cells, and provides a theoretical basis for disclosing a new mechanism of anti-gastric cancer activity of the C21 steroid.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides an application of a C21 steroid in preparing a medicine for promoting cancer cell apoptosis, wherein the C21 steroid inhibits the proliferation of cancer cells by inhibiting autophagy to promote the apoptosis.

Preferably, the C21 steroid promotes the development of the autophagy pathway by activating AKT.

Preferably, the C21 steroid is glaucescent fissistigma root extract.

Preferably, the cancer cell is a gastric cancer cell.

The invention also provides application of the C21 steroid in preparation of a medicine for treating gastric cancer, wherein the C21 steroid is a glaucescent fissistigma root extract, and the C21 steroid inhibits proliferation of gastric cancer cells by inhibiting autophagy and promoting apoptosis.

The invention discloses the following technical effects:

since activation of the PI3K/Akt pathway has been demonstrated in many types of tumors, it contributes to the proliferation and survival of tumor cells. In the invention, experiments verify that the BGC-823 and AGS cells treated with C21 steroid have a significant reduction in p-AKT, indicating that C21 steroid may induce apoptosis through PTEN-AKT signaling pathway. And the inventor proves in previous research that the cochinchina sylvestris extract C21 steroid can induce the apoptosis of liver cancer cells through a PTEN/AKT/mTOR signaling pathway. The PI3K/Akt/mTOR pathway has been shown to play a negative role in regulating autophagy, its decline actually activates autophagy; it has also been found that an autophagy-related protein (ATG) plays a particular role in regulating the formation of autophagosomes encoded by autophagy-related genes. In the invention, the autophagy-related proteins ATG5 and Beclin-1 are increased when C21 steroid induces autophagy and are reduced when autophagy inhibition occurs, further indicating that the C21 steroid can activate autophagy in gastric cancer cells, and indicating that the C21 steroid can induce protective autophagy in gastric cancer cells by inhibiting PI3K/Akt/mTOR signaling pathway. The invention shows that the C21 steroid can inhibit gastric cancer cell proliferation, induce gastric cancer cell apoptosis and autophagy; in particular, C21 steroid enhances gastric cancer apoptosis by inhibiting autophagy, partly due to activation of AKT, and thus C21 steroid will be a new candidate for the treatment of gastric cancer, extracted from natural compounds.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the effect of C21 steroid in inhibiting gastric cancer cell proliferation; a-C shows that C21 steroids at different concentrations inhibit BGC-823, SGC-7901 and AGS cell proliferation respectively: treating gastric cancer cells with a C21 steroid (0, 40, 80, 160. mu.g/mL, analyzing real-time cell detector results with Prism software from GraphPad. treating gastric cancer cells with a C21 steroid (0, 20, 40, 80, 160, 320. mu.g/mL), analyzing MTS results with Prism software from GraphPad. P <0.05, vs NC;

FIG. 2 is a graph showing induction of BGC823 and AGS cell cycle arrest and apoptosis in gastric cells treated with 0, 80 μ g/mL and 160 μ g/mL C21 steroids for 24 h; a: after staining with staining buffer a and reagent B, cell count (%) was determined by flow cytometry; b, cell cycle statistical chart; c: after being stained with Annexin-V-Fluorescein Isothiocyanate (FITC) and PI, the apoptosis rate was measured by flow cytometry; the lower left corner shows normal rate, the lower right corner early apoptosis, the upper right corner late apoptosis; d: a histogram of apoptosis rate; e: western blot analysis to determine clear-PARP protein levels, with β -actin as control; f: statistics of the expression rate of cleaned-PARP; p <0.05, vs NC;

figure 3 shows that C21 steroid treatment promotes gastric autophagy; a: co-localization of green fluorescent protein-LC 3 (green) was observed by confocal microscopy after 24h treatment with C21 steroid (100. mu.g/mL) in the absence or presence of CQ (20. mu.M); b: LC3 protein levels were determined by western blot analysis after exposing cells to C21 steroid (120. mu.g/mL) and CQ (20. mu.M) for 24h, with GAPDH as a control; c: quantifying expression rate statistics for LC 3-II; p <0.05, vs NC;

fig. 4 is the promotion of apoptosis by C21 steroid in combination with CQ; a: after being stained with Annexin-V-FITC and PI, the apoptosis rate is measured by a flow cytometer; the lower left corner is the normal ratio, the lower right corner is early apoptosis, the upper right corner is late apoptosis; b: quantifying the apoptosis percentage respectively; c: after DCFH-DA staining, quantitatively determining ROS level; a-b: after exposing the cells to C21 steroid (120. mu.g/mL) and CQ (20. mu.M) for 24h, the autophagy-related proteins were analyzed using western blot with GAPDH as control; c: quantifying the expression rate of Beclin-1; d: quantifying the expression rate of ATG-5; e: quantifying the expression rate of BCL-2; p <0.05, vs NC;

fig. 5 is a C21 steroid interfering with autophagy through the AKT signaling pathway; a-b: after BGC-823 and AGS cells were exposed to C21 steroid (120. mu.g/mL) and CQ (20. mu.M) for 24h, respectively, proteins associated with apoptosis were determined by Western blot analysis with GAPDH as a control; c: quantifying the expression rate of Beclin-1; d: quantifying the expression rate of ATG-5; e: quantifying the expression rate of p-AKT; p <0.05, vs NC.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

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 invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, 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 invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Materials and methods

Fetal Bovine Serum (FBS) and Roswell Park clinical Institute 1640 Medium RPMI1640 was purchased from HyClone.

C21 steroids from glaucescent fissistigma root extract were provided by the institute of medicine laboratory of university of industry, zhejiang.

The GFP-LC3 plasmid was provided by the clinical laboratory of the people's hospital in Zhejiang province.

3- (45-dimethylthiozol-2-yl) -5- (3-carboxymethyxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium inner salt (MTS) was purchased from PROMEGA.

Chloroquine diphosphate was purchased from Sangon Biotech.

BGC-823, SGC-7901 and AGS cells-human gastric cancer cells-from the clinical laboratory in Min Hospital, Zhejiang province. All cells were cultured in Roswell Park clinical Institute medium 1640(RPMI1640, HyClone) supplemented with 10% fetal bovine serum (FBS, HyClone). All cells were cultured at 37 ℃ with 5% CO₂The incubator of (1).