CN106074567A - Cucurbitacin B application in preparing the carcinous inhibitory factor inhibitors of PP2A - Google Patents

Abstract

The invention belongs to the technical field of medicine, and in particular relates to the application of cucurbitacin B, a tetracyclic triterpenoid compound isolated and extracted from Cucurbitaceae plants, as an inhibitor of protein phosphatase 2A cancer inhibitory factor. In the present invention, cucurbitacin B, as the main component in the protein phosphatase 2A cancer inhibitor inhibitor, can significantly inhibit the expression of protein phosphatase 2A cancer inhibitor, and can be administered alone or in combination, including radiotherapy, chemotherapy, surgery Treatment, other growth factor inhibitors and sex hormone inhibitors, and any combination of tumor treatment drugs and methods.

Description

Application of Cucurbitacin B in Preparation of Protein Phosphatase 2A Cancer Inhibitor Inhibitor

technical field

The invention belongs to the technical field of medicine, and in particular relates to the application of cucurbitacin B in the preparation of inhibitors of protein phosphatase 2A cancer inhibitors.

Background technique

Tumors, especially malignant tumors, are a major disease that threatens human health. Tumor treatment is a major medical problem facing mankind today. Tumors are often caused by gain-of-function mutations in oncogenes and loss-of-function mutations in tumor suppressor genes. Mutations in these genes often lead to tumor cell escape from apoptosis, insensitivity to normal anti-growth signals, acquisition of self-sufficient growth signals and unlimited replication potential, as well as the ability of tissue invasion and metastasis, resulting in recurrence and drug resistance. Therefore, tumors are actually diseases in which cell signaling occurs abnormally. Finding compounds that specifically bind and inhibit the activity of factors that play a key role in the development of tumors has positive significance for tumor mechanism research and treatment.

Protein phosphatase 2A cancer inhibitor (cancerous inhibitor of protein phosphatase 2A, CIP2A) is an endogenous cancer inhibitor of protein phosphatase 2A (protein phosphatase 2A, PP2A). The constitutive phosphorylation of c-Myc serine 62 stabilizes the expression of c-Myc protein. At the same time, as a protein serine/threonine phosphatase, PP2A acts as a tumor suppressor in tumor cells. As an upstream factor of Akt and ERK, PP2A can directly dephosphorylate Akt and ERK. It has been reported that in many cancers, the activity of PP2A is inhibited, which is closely related to the malignant transformation of cancer cells. The inhibition of PP2A activity in tumor cells is mostly due to the overexpression of CIP2A in tumor cells. In recent years, the role of CIP2A in various solid tumors including lung cancer, gastric cancer, and breast cancer and blood system tumors has been continuously discovered. Tumor malignant transformation, recurrence and drug resistance. It has been reported that in cancer cells, reactivation of PP2A activity by inhibiting CIP2A can significantly inhibit tumor growth and malignant transformation. Therefore, screening compounds that directly target CIP2A is of great significance for improving the efficacy of tumor therapy.

Contents of the invention

The invention uses cucurbitacin B to inhibit CIP2A, thereby activating the activity of its substrate tumor suppressor protein PP2A to achieve the purpose of individualized treatment.

In the present invention, cucurbitacin B can be any pharmaceutically acceptable cucurbitacin B salt, and the formed CIP2A inhibitor with cucurbitacin B or its salt as the main component can be any pharmaceutically acceptable dosage form , the dosage of cucurbitacin B can be any pharmacologically acceptable dosage.

The invention is used for treating stomach cancer, breast cancer, lung cancer and neuroglioma. , can be administered alone or in combination, including combined application with radiotherapy, chemotherapy, surgery, other growth factor inhibitors and sex hormone inhibitors, and any drugs and methods for treating tumors. In particular, the combination of cucurbitacin B and cisplatin can effectively treat gastric cancer, lung cancer and glioma.

Cucurbitacin B can significantly inhibit the expression of CIP2A in cells and animal tumor-bearing models, and has no obvious toxic and side effects on other normal tissues. At the same time, it has the advantages of moderate dose, significant curative effect, and clear targets. It has broad anti-cancer application prospects in clinic.

Description of drawings

Figure 1 shows the effect of cucurbitacin B of the present invention on the growth of gastric cancer, breast cancer, lung cancer and glioma cells and normal cells.

Fig. 2 is a diagram showing the detection of CIP2A expression levels in gastric cancer, breast cancer, lung cancer and glioma cells inhibited by cucurbitacin B of the present invention.

Fig. 3 is a graph showing the activity detection of CIP2A substrate PP2A activated by cucurbitacin B of the present invention in gastric cancer, breast cancer, lung cancer and glioma cells.

Fig. 4 is a comparison chart of the synergistic anti-gastric cancer, breast cancer, lung cancer and glioma cells of the combined application of cucurbitacin B and cisplatin of the present invention.

detailed description

The present invention is further explained below in conjunction with specific examples. The main materials used in the embodiment are as follows:

Cucurbitacin B: purchased from Shanghai Yuanye Biotechnology Co., Ltd., the purity is greater than 95% as determined by high performance liquid chromatography (HPLC). Use dimethyl sulfoxide (DMSO) to prepare a 100mmol/L stock solution, freeze at -20°C, and dilute to the working concentration with the medium each time it is used.

Cell lines: All cell lines used in the examples were purchased from ATCC or the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Antibody: CIP2A, antibody was purchased from Santa Cruz, USA, PP2A, GAPDH antibody was purchased from Cell Signaling Technology, USA. PP2A protein phosphatase activity assay kit was purchased from Upstate, USA.

Example 1 Cucurbitacin B inhibits gastric cancer, breast cancer, lung cancer and glioma cell growth

Various types of cancer cell lines SGC7901 (gastric cancer), MCF-7/Adr (breast cancer), NCI-H1975 (lung cancer), and DBTRG (glioma) cells in logarithmic growth phase and human normal bronchial Epithelial cells 16-HBE, human normal gastric mucosal epithelial cells GES-1 were inoculated in 96-well plate (inoculate about 5000 cells per well, 90 μl medium), cultured (37°C, 5% _CO2 incubator) for 24 hours before use Cucurbitacin B (Cucurbitacin B, CuB) with different concentration gradients (10-1200nmol/L) treated five kinds of cells. After culturing for 20 and 44 hours, add 10 μl of 5 mg/ml 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) solution to each well, continue Incubate for 4 hours. After the reaction was terminated, the culture medium was sucked off, 150 μl dimethyl sulfoxide was added to each well, shaken at a low speed to fully dissolve, and then the absorbance value at 490 nm (OD490) was measured with a microplate reader. According to the results of growth inhibition rate, the 24-hour half maximal inhibitory concentration (IC ₅₀ ) of CuB to six kinds of cells was calculated. The specific IC ₅₀ value is shown in Figure 1F. The results showed that CuB can inhibit the proliferation of many different types of cancer cells (Figure 1A- 1D), and relatively less toxic to normal 16-HBE and GES-1 (Fig. 1E).

Example 2 Cucurbitacin B inhibits the expression of CIP2A protein in gastric cancer, breast cancer, lung cancer and glioma cells.

The specific implementation method is as follows:

Various types of cancer cell lines SGC7901 (gastric cancer), MCF-7/Adr (breast cancer), NCI-H1975 (lung cancer) and DBTRG (glioma) cells were seeded in 6-well plates at a certain density, 24 After 1 hour, when the degree of cell fusion reached 80%, SGC7901 cells were treated with different concentrations of CuB (0nmol/L, 25nmol/L, 50nmol/L, 100nmol/L), MCF-7/Adr (0nmol/L, 100nmol/L L, 200nmol/L, 400nmol/L), NCI-H1975 cells (0nmol/L, 100nmol/L, 200nmol/L, 300nmol/L), DBTRG cells (0nmol/L, 100nmol/L, 200nmol/L, 400nmol/L L), 24 hours later, the cells were lysed with 1×SDS Loading Buffer to extract the total protein, and the same amount of protein was used for Western Blot immunoblotting, and anti-CIP2A and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibodies were used to detect Western blotting was performed to detect the changes in their expression under drug treatment. The results are shown in Figure 2, gastric cancer cell SGC7901 (Figure 2A), breast cancer cell MCF-7/Adr (Figure 2B), lung cancer cell NCI-H1975 (Figure 2C), and glioma cell DBTRG (Figure 2D) , CuB can dose-dependently down-regulate the expression of CIP2A protein.

Example 3 Cucurbitacin B inhibits the phosphatase activity of the substrate protein PP2A of CIP2A in gastric cancer, breast cancer, lung cancer and glioma cells

The specific implementation method is as follows:

Various types of cancer cell lines SGC7901 (gastric cancer), MCF-7/Adr (breast cancer), NCI-H1975 (lung cancer) and DBTRG (glioma) cells were inoculated on a 10 cm culture dish according to a certain density, 24 Hours later, when the degree of cell fusion reached about 70%-80%, the SGC7901 cells (0nmol/L, 25nmol/L, 50nmol/L, 100nmol/L), MCF-7/Adr (0nmol/L) were treated with different concentrations of CuB respectively. , 100nmol/L, 200nmol/L, 400nmol/L), NCI-H1975 cells (0nmol/L, 100nmol/L, 200nmol/L, 300nmol/L), DBTRG cells (0nmol/L, 100nmol/L, 200nmol/L , 400nmol/L), after 24 hours, four kinds of cells treated with different concentrations of CuB were lysed with RIPA lysate, and the protein solution was stored at -20°C. Take 100 μg protein, respectively, and incubate with 4 μg PP2A antibody, and incubate at 4°C for 10 hours. 40 μl of protein A agarose beads (proteinA agarose beads) were added to the mixture, and incubated at 4° C. for 2 hours. The beads were collected by centrifugation, washed three times with pre-cooled 700 μl PBS, and then washed once with 500 μl Ser/ThrAssay Buffer. Next, add 750 mmol/L phosphopeptide and incubate at 30° C. for 10 minutes with constant stirring. In the next step, 100 μl of Malachite Green Phosphate Detection Solution (Malachite Green Phosphate Detection Solution) was added, and after mixing, the absorbance was detected by a multifunctional microplate reader (Bio-Tek) at a wavelength of 650 nm. As shown in FIG. 3 , cucurbitacin B inhibits the phosphatase activity of CIP2A substrate protein PP2A in gastric cancer, breast cancer, lung cancer and glioma cells. The experimental results showed that with the increase of the concentration, the activity of PP2A enzyme showed an obvious upward trend. * indicates a significant difference compared with the control group (*, p<0.05; **, p<0.01).

Example 4 Detection experiment of cucurbitacin B and cisplatin synergistically inhibiting gastric cancer, lung cancer and glioma cells

After pre-attaching various types of cancer cell lines SGC7901 (gastric cancer), MCF-7/Adr (breast cancer), NCI-H1975 (lung cancer) and DBTRG (glioma) cells for 24 hours, they were treated with different concentrations of CuB (0nmol/L, 25nmol/L, 50nmol/L) combined with cisplatin (0μmol/L, 10μmol/L, 20μmol/L) to treat BGC823 cells; with different concentrations of CuB (0M, 50nmol/L, 100nmol/L) Cisplatin (0 M, 10 μmol/L, 20 μmol/L) was used to treat NCI-H1975 cells; different concentrations of CuB (0 μmol/L, 50 nmol/L, 100 nmol/L) combined with cisplatin (0 μmol/L, 10 μmol/L, 20 μmol/L L) Treat the glioma U251 cells, add MTT detection reagent and incubate for another 4 hours after 44 hours, then measure the absorbance value (OD490) at 490nmol/L with a microplate reader. The results are shown in Figure 4, cucurbitacin B combined with cisplatin treatment can significantly reduce the OD490 of gastric cancer, lung cancer and glioma cells, showing a significant synergistic effect, but cucurbitacin B combined with cisplatin can not significantly reduce the OD490 of breast cancer cells. OD490. Cucurbitacin B and cisplatin synergistically inhibit gastric cancer, lung cancer and glioma cells.

The above detailed description is a specific description of a feasible embodiment of the invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or change that does not deviate from the present invention shall be included in the patent scope of the present invention.

In addition, those skilled in the art can also make various modifications, additions and substitutions in other forms and details within the scope and spirit disclosed in the claims of the present invention. Certainly, the various modifications, additions, substitutions and other changes made according to the spirit of the present invention shall all be included within the scope of protection claimed by the present invention.

Claims (7)

1. the carcinous inhibitory factor inhibitors of PP2A, it is characterised in that use Cucurbitacin B suppression phosphoprotein phosphatase The carcinous inhibitive factor of 2A.

The carcinous inhibitory factor inhibitors of PP2A the most according to claim 1, it is characterised in that Cucurbitacin B is permissible For Cucurbitacin B salt acceptable on any pharmacotherapeutics.

The carcinous inhibitory factor inhibitors of PP2A the most according to claim 1, it is characterised in that Cucurbitacin B is permissible For dosage form acceptable on any pharmacotherapeutics.

The carcinous inhibitory factor inhibitors of PP2A the most according to claim 1, it is characterised in that: Cucurbitacin B and Its analog can be acceptable dosage on any pharmacotherapeutics.

The carcinous inhibitory factor inhibitors of PP2A the most according to claim 1, it is characterised in that be used for treating stomach Cancer and glioma.

The carcinous inhibitory factor inhibitors of PP2A the most according to claim 1, it is characterised in that can be individually dosed Or therapeutic alliance, including with radiotherapy, chemotherapy, operative treatment, other growth factor receptor inhibitors and gonadal hormone inhibitor, Yi Jiren What anti-tumor medicine and use in conjunction of method.

Combinational therapeutic methods the most according to claim 6, it is characterised in that Cucurbitacin B can be with cisplatin combined use.